US 2011 0014129A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2011/0014129 A1 ZabOW et al. (43) Pub. Date: Jan. 20, 2011
(54) MAGNETIC MICROSTRUCTURES FOR (60) Provisional application No. 61/166,610, filed on Apr. MAGNETIC RESONANCE MAGING 3, 2009.
(76) Inventors: Gary Zabow, Boulder, CO (US); Publication Classification Stephen Dodd, Rockville, MO (US); Alan Korelsky, Bethesda, (51) Int. Cl. MO (US); John M. Moreland, A61R 49/18 (2006.01) Louisville, CO (US) A6B 5/55 (2006.01) GOIR 33/44 (2006.01) Correspondence Address: National Institutes of Health (52) U.S. Cl...... 424/9.34; 424/9.3; 600/419; 324/307 c/o Polsinelli Shugart PC (57) ABSTRACT 161 N. Clark Street, Suite 4200 Chicago, IL 60601 (US) The present invention relates to a magnetic resonance struc ture with a cavity or a reserved space that provides contrast (21) Appl. No.: 12/753,689 and the additional ability to frequency-shift the spectral sig nature of the NMR-susceptible nuclei such as water protons (22) Filed: Apr. 2, 2010 by a discrete and controllable characteristic frequency shift that is unique to each MRS design. The invention also relates Related U.S. Application Data to nearly uniform solid magnetic resonance T contrast (63) Continuation-in-part of application No. PCT/US09/ agents that have a significantly higher magnetic moment 41142, filed on Apr. 20, 2009. compared to similarly-sized existing MRI contrast agents.
1100
4. Patent Application Publication Jan. 20, 2011 Sheet 1 of 47 US 2011/0014129 A1
114 Patent Application Publication Jan. 20, 2011 Sheet 2 of 47 US 2011/0014129 A1
………………××××××=*******
FIG 2 Patent Application Publication Jan. 20, 2011 Sheet 3 of 47 US 2011/0014129 A1
NOTOSCAE
12 SBSRAE
TTEM
NSS GOD
N NCKE
ZCOPPER SE POTORESS HARDBAKED) POORESS is 88 EPOXY Patent Application Publication Jan. 20, 2011 Sheet 4 of 47 US 2011/0014129 A1
OOSCE YY SUBS RAE YY AN O SS NCKE. COPPER pHCORESST Patent Application Publication Jan. 20, 2011 Sheet 5 of 47 US 2011/0014129 A1
NO ESCAL SESTRATE
A: NYS GO KE
CPR
4 (4 4 E TORESS
777 HARDBAKED :::::: S3EFOXY
FIG. 4B Patent Application Publication Jan. 20, 2011 Sheet 6 of 47 US 2011/0014129 A1
:3. ::8: sess OTC SCAL
YY SSRT
FAN
Ayyyy year XXXIX's
COPPER Egg PHOORESIST (LIFT-OFF s: S3EPOXY Patent Application Publication Jan. 20, 2011 Sheet 7 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 8 of 47 US 2011/0014129 A1
. 2 APPEED FELD 347 Air FIG. 7
x: Patent Application Publication Jan. 20, 2011 Sheet 9 of 47 US 2011/0014129 A1
FIG 9
SSSSSSS SSSS& SS i.S. FIG 10 Patent Application Publication Jan. 20, 2011 Sheet 10 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 11 of 47 US 2011/0014129 A1
ass
3.
5 4::: 3 DOFREQUENCY SHIFT kHz FIG. 12
e
v THEORY -ili 4. 20 FREQUENCY SHIFT kHz FIG. 13 Patent Application Publication Jan. 20, 2011 Sheet 12 of 47 US 2011/0014129 A1
100
s
FREQUENCY SHIFT kHz FIG. 14
s
-- Rs.25am 80 -- Rs. 5pm 500 400 360 to -160 FREQUENCY SHIFT kHz FIG. 15 Patent Application Publication Jan. 20, 2011 Sheet 13 of 47 US 2011/0014129 A1
FREQUENCY SHIFT . FIG 16
FIG. 17 Patent Application Publication Jan. 20, 2011 Sheet 14 of 47 US 2011/0014129 A1
FIG. 18B FIG. 18C FIG. 18A
5: 88: 'sitS. sh: FIG. 18D FIG. 18E
3,704
8 300 RE li: SH:8 FIG. 19A FIG. 19B Patent Application Publication Jan. 20, 2011 Sheet 15 of 47 US 2011/0014129 A1
FIG. 20A
FIG. 20B Patent Application Publication Jan. 20, 2011 Sheet 16 of 47 US 2011/0014129 A1
FIG 21A FIG 21D
N
\SSSSSS
FIG 21E
FIG 21 F Patent Application Publication Jan. 20, 2011 Sheet 17 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 18 of 47 US 2011/0014129 A1
e ,
5
FREQUENCY SHIFT kHz FIG. 23A
-1250 750
FIG.FREQUENCY 23B SHIFT kHz Patent Application Publication Jan. 20, 2011 Sheet 19 of 47 US 2011/0014129 A1
FREQUENCY SHIFTHz FIG. 23C
so FREQUENCY SHIFT kHz FIG. 23D Patent Application Publication Jan. 20, 2011 Sheet 20 of 47 US 2011/0014129 A1
FIG. 24 A Patent Application Publication Jan. 20, 2011 Sheet 21 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 22 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 23 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 24 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 25 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 26 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 27 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 28 of 47 US 2011/0014129 A1
xx xxxx.x re. Patent Application Publication Jan. 20, 2011 Sheet 29 of 47 US 2011/0014129 A1
FIG. 34 Patent Application Publication Jan. 20, 2011 Sheet 30 of 47 US 2011/0014129 A1
*:::::s: Fr88.8. shire FIG. 35 Patent Application Publication Jan. 20, 2011 Sheet 31 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 32 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 33 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 34 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 35 of 47 US 2011/0014129 A1
FIG. 40 Patent Application Publication Jan. 20, 2011 Sheet 36 of 47 US 2011/0014129 A1
Patent Application Publication Jan. 20, 2011 Sheet 37 of 47 US 2011/0014129 A1 ae?:??: ~ ~ :%??????????????????????????????? ae
. .
? Patent Application Publication Jan. 20, 2011 Sheet 39 of 47 US 2011/0014129 A1 Patent Application Publication Jan. 20, 2011 Sheet 40 of 47 US 2011/0014129 A1 Patent Application Publication Jan. 20, 2011 Sheet 41 of 47 US 2011/0014129 A1 A ki:k: t 88: C : life: Patent Application Publication Jan. 20, 2011 Sheet 42 of 47 US 2011/0014129 A1 FIG. 47 Patent Application Publication Jan. 20, 2011 Sheet 43 of 47 US 2011/0014129 A1 FIG. 48 Patent Application Publication Jan. 20, 2011 Sheet 44 of 47 US 2011/0014129 A1 Sists: Experiter iron 2 a. S. oST, n. i. SS s Nike """", i.e. S San : 2..w...see l sex...xxxx..., s 8.- :"; i. SS S St. FIG. 49 Patent Application Publication Jan. 20, 2011 Sheet 45 of 47 US 2011/0014129 A1 50 sm 100 urn 23 ms s 8X to:s' Patent Application Publication Jan. 20, 2011 Sheet 46 of 47 US 2011/0014129 A1 S S S · 8 8 & & Patent Application Publication Jan. 20, 2011 Sheet 47 of 47 US 2011/0014129 A1 FIG. 53 US 2011/0014129 A1 Jan. 20, 2011 MAGNETIC MICROSTRUCTURES FOR and therefore prevented in vivo tracking of single cells from MAGNETC RESONANCE MAGING becoming routine. Accordingly, there is a need in the art for an improved contrast agent. CROSS-REFERENCE TO RELATED SUMMARY OF THE INVENTION APPLICATION 0006. The present invention is directed to a magnetic reso 0001. This is a continuation-in-part of International Appli nance contrast agent consisting essentially of a plurality of cation No. PCT/US2009/041142, filed on Apr. 20, 2009, disks of uniform size and magnetic moment, wherein the which claims the benefit of U.S. Provisional Patent Applica disks consist essentially of a single magnetic material. Each tion No. 61/071,263, filed Apr. 18, 2008, the contents of disk may have magnetic moment from about 10' A m to which are incorporated herein by reference. This also claims about 10' A'm and vary in size by less than about 5% of the average size of the plurality of disks. Each disk may also vary the benefit of U.S. Provisional Patent Application No. 61/166, in magnetic moment by less than about 5% of the average 160, filed Apr. 3, 2009, the contents of which are hereby magnetic moment of the plurality of disks. The magnetic incorporated by reference. material of the magnetic resonance contrast agent may com prise a ferromagnetic, paramagnetic, Superparamagnetic, FIELD OF INVENTION magnetic alloy, or a magnetic compound. The magnetic reso nance contrastagent may further comprises a coating selected 0002 The present invention relates to magnetic resonance from an oxidation barrier, a corrosion barrier, a mechanical imaging contrast agents and methods of magnetic resonance strengthening layer, a non-toxic coating, a biologically inert imaging. In particular, the present invention relates to mag coating, a coating to facilitate common bioconjugation pro netic resonance structures used as magnetic resonance imag tocols, a cell-specific antibody or ligand coating, and combi ing contrast agents and multiplexed magnetic resonance nations thereof. The magnetic resonance contrast agent may imaging methods. be a disk shape having a disk diameterranging from about 0.5 um to about 10 Lum and a disk thickness ranging from about 0.5um to about 10 um. BACKGROUND 0007. The present invention is also directed to a method of Super-resolution tracking of a magnetic resonance visualiza 0003 Biotechnology and biomedical research have ben tion contrast agent consisting essentially of a plurality of efited from the introduction of a variety of specialized nano disks, wherein the disks consist essentially of a single mag particles whose well-defined, optically distinguishable signa netic or paramagnetic material. The method may comprise (a) tures enable simultaneous tracking of numerous biological distributing the magnetic resonance visualization contrast indicators. Optically based labels such as colored fluoro agent within a sample such that each individual disk is spa phores, multi-spectral semiconductor quantum dots, and tially separated from all other disks; (b) performing magnetic metallic nanoparticles can be used for multifunctional encod resonance visualization of the sample to obtain an magnetic ing, and biomolecular sensing and tracking. However, these resonance visualization image comprising a plurality of VOX optically based labels can probe only so far beneath most els, wherein each Voxel comprises a pixel, each pixel having Surfaces. a pixel intensity; (c) analyzing the magnetic resonance image 0004 Contrast agents used in magnetic resonance may to locate each pixel that is darker than a background pixel intensity resulting from a disk somewhere within a corre probe far below most tissue surfaces. Equivalent multiplexing sponding Voxel, determining if any of the darkest voxels are capabilities are largely absent in the field of magnetic reso situated in a contiguous group (d) determining the pixel inten nance imaging (MRI). MRI cell tracking is based on the sity of the darkest voxel; assigning the location of the contrast magnetically dephased signal from the fluid Surrounding cells agent to the darkest voxels in each contiguous group and (e) labeled with many superparamagnetic iron oxide (SPIO) determining the location of each disk within its correspond nanoparticles, or dendrimers, or individual micrometer-sized ing Voxel by comparing the intensity of the Voxel in each particles of iron oxide (MPIO). The continuous spatial decay contiguous group to the intensity of the darkest voxel in the of the external fields Surrounding these magnetizable par contiguous group. ticles, or any other magnetizable particles, imposes a continu 0008. The present invention is also directed to a method of ous range of Larmor frequencies that broadens the water non-invasively monitoring at least one characteristic of blood hydrogen proton line, obscuring any distinction between dif flow through a stent device situated within a blood vessel of a ferent types of magnetic particles that might specifically label living Subject. The method may comprise (a) providing a stent different types of cells and as a consequence provide only a device comprising an MRS, wherein the MRS induces a monochrome contrast. Accordingly, there is a need in the art known NMR shift in a NMR-susceptible nucleus when to distinguish with magnetic resonance (MR) between differ exposed to an excitatory electromagnetic pulse delivered at a ent cell types, at the single-cell level, for application in cel corresponding resonance frequency; (b) situating the stent lular biology, and early disease detection and diagnosis. device within the blood vessel of the living subject; (b) expos 0005. Alternatively, cellular tracking and labeling by ing the stent device to at least one or more excitatory electro strong magnetic resonance T agents can also be used for magnetic pulses delivered at the corresponding resonance labeling to provide a strong monochrome contrast to cellular frequency to create a volume of spin-labeled blood mol components. T. contrast agents such as nanoscale Super ecules; (c) obtaining nuclear magnetic resonance data of a paramagnetic particles of iron oxide (SPIOs) and their volume of blood flowing downstream of the stent device; and, micrometer-sized equivalents (MPIOs) can only be used in (d) analyzing the magnetic resonance image data to locate the limited amounts in a cell without compromising its viability volume of spin-labeled blood molecules using the known US 2011/0014129 A1 Jan. 20, 2011 NMR shift. At least one characteristic of blood flow of the 0017 FIG. 4C is an illustration of a method of manufac method may comprise mass flow rate, Volume flow rate, and turing magnetic series of intermediate structures produced flow speed. In addition, the characteristic of blood flow may during still another embodiment of a method of manufactur be compared to a baseline characteristic of blood flow to ing magnetic resonance microstructures. determine the presence or absence of occlusions within the 0018 FIG. 5 is an illustration of an embodiment of a stent device. The NMR shift of the spin-labeled blood mol magnetic resonance identity system. ecules may be compared to a baseline NMR shift to determine 0019 FIG. 6 is a graph of the calculated particle volume if any deformation in the shape of the stent device has fraction that falls within a bandwidth, 8c), about the particle's occurred and each different NMR shift is assigned a different frequency shift, Act) for an embodiment of a magnetic reso color on a color scale. nance microstructure. 0009. The present invention may also be directed to an 0020 FIG. 7 is a graph of an alternating-gradient magne magnetic resonance contrast agent comprising a magnetic tometer hysteresis curve for an embodiment of a dual-disk material forming a reserved space that is connected to a magnetic resonance structure. near-field volume, wherein the magnetic material produces a 0021 FIG. 8 is a scanning electron micrographs (SEM) substantially uniform magnetic field within the reserved image of an embodiment of a dual-disk magnetic resonance space, and wherein the uniform magnetic field has a magni Structure. tude that is substantially different than a background mag (0022 FIG. 9 is a SEM image of another embodiment of a netic field within the near-field volume. dual-disk magnetic resonance structure. 0010. The present invention may also be directed to a method of using two or more MRS, wherein each MRS (0023 FIG. 10 is another SEM image of yet another induces a known NMR shift in a NMR-susceptible nucleus embodiment of a dual-disk magnetic resonance structure. when exposed to an excitatory electromagnetic pulse deliv 0024 FIG. 11 is a graph of the Z-spectra produced using ered at a corresponding resonance frequency, wherein each of three embodiments of the dual-disk magnetic microstruc the known NMR shifts, and each of the corresponding reso tures. nance frequencies are different for each of the two or more 0025 FIG. 12 is a graph of the Fourier transformed spin MRS. The method may comprise (a) distributing the two or echo signal generated from direct MRI imaging from an more MRS within a sample; (b) exposing the two or more embodiment of the dual-disk magnetic resonance structures. MRS within the sample to excitatory electromagnetic pulses 0026 FIG. 13 is a graph showing the z-spectra produced delivered at each of the two or more corresponding resonance by an embodiment of the dual disk magnetic resonance struc frequencies; (c) obtaining nuclear magnetic resonance data ture using difference delays (AT), between off-resonant JL/2 after each excitatory electromagnetic pulse; and, (d) using the pulses. known NMR shifts to determine the identity of each of the 0027 FIG. 14 is a graph showing the z-spectra produced two or more MRS. Each particular MRS of the method may by an embodiment of the dual-disk magnetic resonance struc be targeted toward a particular tissue or cell. The MRS tar ture measured at three different field-strengths. geted to a particular cell may be bound to the cellata cell wall 0028 FIG. 15 is a graph showing the z-spectra produced or cell membrane. by two embodiments of the dual-disk magnetic resonance structure having difference disk radii. BRIEF DESCRIPTION OF THE DRAWINGS 0029 FIG. 16 is a graph showing a map of the Z-spectra of 0011 Additional features of this invention are provided in numerous embodiments of the dual-disk magnetic resonance the following detailed description of various embodiments of structures having different disk thicknesses (h). the invention with reference to the drawings. Furthermore, 0030 FIG. 17A is an image of a high tilt angle SEM the above-discussed and other attendant advantages of the showing a square array of an embodiment of the dual-disk present invention will become better understood by reference magnetic resonance particle; in which part of the particles to the detailed description when taken in conjunction with the have filled interior regions. accompanying drawings, in which: 0031 FIG. 17B is an MRI image of the dual-disk magnetic 0012 FIG. 1 is an illustration of an embodiment of a resonance particles shown in 17A. dual-disk magnetic resonance structure. 0032 FIGS. 18A is an illustration of an embodiment of a 0013 FIG. 2 is a contour graph showing the calculated hollow cylinder structure. magnitude of a magnetic field throughout a plane oriented 0033 FIG. 18B is a graph showing the calculate magnetic between the disks of an embodiment of a dual-disk magnetic field magnitude profile in a mid-plane through an embodi reSOnance Structure. ment of a hollow cylinder magnetic resonance structure. 0014 FIG. 3 is an illustration of a method of manufactur 0034 FIG. 18C is a graph showing the calculated mag ing magnetic series of intermediate structures produced dur netic field profile in a perpendicularly oriented mid-plane ing an embodiment of a method of manufacturing magnetic through an embodiment of a hollow cylinder magnetic reso resonance microstructures. nance Structure. 0.015 FIG. 4A is an illustration of a method of manufac 0035 FIG. 18D is a graph showing a histogram recording turing magnetic series of intermediate structures produced of the estimated frequency shifts in the Volume Surrounding during another embodiment of a method of manufacturing an embodiment of a hollow cylinder magnetic resonance magnetic resonance microstructures. Structure. 0016 FIG. 4B is an illustration of a method of manufac 0036 FIG. 18.E is a graph showing the calculated internal turing magnetic series of intermediate structures produced Volume fraction of an embodiment of a hollow cylinder mag duringyetanother embodiment of a method of manufacturing netic resonance structure falling within a bandwidth Öc) of a magnetic resonance microstructures. central frequency shift Act). US 2011/0014129 A1 Jan. 20, 2011 0037 FIG. 19A is a graph showing the spectron of numer 0.058 FIG. 27 is a drawing showing a flow tagging appli ous embodiments of hollow cylinder magnetic resonance cation of an embodiment of a hollow cylindrical magnetic structures having different length to diameter rating (L/2p). resonance structure at two different flow speeds. 0038 FIG. 19B is a graph showing the spectra of numer 0059 FIGS. 28-31 show experimental results for an ous embodiments of hollow cylinder magnetic resonance embodiment corresponding to FIG. 27. structures having different degrees of wall thickness variation 0060 FIG. 32 is a graph showing the Z-spectra of an (At/t). embodiment of a dual-disk magnetic resonance structure in 0039 FIG. 20A is a drawing that illustrates the geometri which one disk is Smaller in radius. cal quantities used in equation X (sputtering equation). 0061 FIG. 33 is a graph showing the Z-spectra of an 0040 FIG. 20B is a drawing showing the calculated side embodiment of a dual-disk magnetic resonance structure in wall coating thicknesses for embodiments of the hollow cyl which one disk is smaller in radius and thicker than the other inder magnetic resonance structure fabricated using cos' O. disk. cos 0, and cos () sputter distributions. 0062 FIG. 34 is a graph showing the Z-spectra of an 0041 FIGS. 21A-21F are drawings illustrating the inter embodiment of a dual-disk magnetic resonance structure in mediate precuts of an embodiment of a fabrication process for which one disk is offset relative to the other disk in a direction hollow cylinder magnetic resonance particles. perpendicular to the applied magnetic field. 0042 FIG. 21A is a drawing showing cylindrical photo 0063 FIG. 35 is a graph showing the Z-spectra of an resist posts atop a gold-titanium coated Substrate. embodiment of a dual-disk magnetic resonance structure in 0043 FIG.21B is a drawing of angled copper evaporation which one disk is offset relative to the other disk in a direction onto the cylindrical photoresist. parallel to the applied magnetic field. 0044 FIG. 21C is a drawing showing magnetic material 0064 FIG. 36 is a graph showing the effect of manufac evaporation. turing variation on the Z-spectra of an embodiment of a dual 0045 FIG. 21D is a drawing showing ion-milling removal disk magnetic resonance structure. of magnetic material and local resputtered coating of the 0065 FIGS. 37A-37F are drawings illustrating the inter photoresist posts. mediate steps of an embodiment of a fabrication process for a 0046 FIG. 21E is a drawing showing copper and photo dual-disk magnetic resonance structure. resist removal. 0.066 FIG. 38 are drawings illustrating the effect of the 0047 FIG.21F is a drawing showing the release of hollow radial distance from the wafer center on the profiles of evapo cylinders magnetic resonance structures by gold-etch or rated lift-off patterned deposits during an embodiment of a ultrasound techniques. fabrication process for a dual-disk magnetic resonance struc 0048 FIG. 22A is an image from a scanning electron ture. micrograph (SEM) of fabricated hollow cylinder magnetic 0067 FIG. 39 is a scanning electron micrograph of a resonance structures produced by an embodiment of a fabri trilayer evaporated nickel-copper-nickel cylindrical stacks cation process. resulting from an embodiment of a fabrication process for a 0049 FIG. 22B is an image from a scanning electron dual-disk magnetic resonance structure. micrograph (ps425 nm) showing an embodiment of a hollow 0068 FIG. 40 is a scanning electron micrograph compar cylinder magnetic resonance structure in the absence of an ing the sizes of each disk in a dual-disk pair resulting from an applied magnetic field (top image) and in the presence of an embodiment of a fabrication process for a dual-disk magnetic applied field (bottom image). reSOnance Structure. 0069 FIGS. 41A-41D are a drawings illustrating an 0050 FIG. 23A is a graph showing the Z-spectra of an embodiment of a fabrication method for a solid high magnetic embodiment of a hollow cylinder magnetic resonance struc moment T. contrast agent. ture having a radius of 1 Jum, a wall thickness of 75 nm. 0070 FIG. 42A-42B are scanning electron micrographs 0051 FIG. 23B is a graph showing the Z-spectra of an showing an embodiment of a solid high magnetic moment embodiment of a hollow cylinder magnetic resonance struc T* contrast agent. ture having a radius of 1 Jum, a wall thickness of 150 nm. 0071 FIG. 43 is a graph showing the effect of transverse 0052 FIG. 23C is a graph showing the Z-spectra of an dephasing on theoretical single Voxel signal intensities during embodiment of a hollow cylinder magnetic resonance struc magnetic resonance imaging of a Solid high magnetic ture having a radius of 425 nm, a wall thickness of 40 nm. moment T. contrast agent using spherical voxel geometry. 0053 FIG. 23D is a graph showing the Z-spectra of an 0072 FIG. 44 is a graph showing the effect of transverse embodiment of a hollow cylinder magnetic resonance struc dephasing on theoretical single Voxel signal intensities during ture having a radius of 450 nm, a wall thickness of 50 nm. magnetic resonance imaging of a Solid high magnetic 0054 FIG. 23E is an MRI image of an array of hollow moment T. contrast agent using cubic voxel geometry. cylinder magnetic resonance structure in which a Subset of the 0073 FIG. 45 is a graph comparing theoretical single hollow cylinders is filled in. Voxel signal intensities from the magnetic resonance image of 0055 FIG.24A is an image of a gradient-echo MRI show a solid high magnetic moment T. contrast agent with and ing hypointense T contrast (dark spots) Surrounding loca without image distortion corrections. tions of embodiments of the hollow cylinder magnetic reso (0074 FIGS. 46A-46D are simulated gradient echo MRI nance Structures. images of an embodiment of a solid high magnetic moment 0056 FIG. 25 is a drawing illustrating another of a stent T* contrast agent made of various magnetic materials. magnetic resonance structure. 0075 FIG. 46A is a simulated gradient echo MRI images 0057 FIG. 26 is a drawing illustrating another embodi taken using 50-um isotropic resolution and a magnetic field ment of a stent magnetic resonance structure. Bo oriented parallel to the MRI image slices. US 2011/0014129 A1 Jan. 20, 2011 0076 FIG. 46B is a simulated gradient echo MRI images 0096 FIG. 49B is a histogram of the experimentally-mea taken using 100-um diameter contrast agent particles using a Sured single Voxel signal intensity for a microfabricated magnetic field Bo oriented parallel to the MRI image slices. nickel contrast agent normalized to the background signal 0077 FIG. 46C is a simulated gradient echo MRI images intensity. taken using 50-um isotropic resolution and a magnetic field 0097 FIG. 49A is a histogram of theoretical single voxel B oriented perpendicular to the MRI image slices. signal intensity for a MPIO contrast agent normalized to the 0078 FIG. 46D is a simulated gradient echo MRI images background signal intensity. taken using 100-um isotropic resolution and a magnetic field 0.098 FIG. 49B is a histogram of the experimentally-mea Bo oriented perpendicular to the MRI image slices. Sured single Voxel signal intensity for a MPIO contrast agent 007.9 FIG. 46A is a simulated gradient echo MRI images normalized to the background signal intensity. taken using 50-um isotropic resolution and a magnetic field (0099 FIGS. 50A-50F are graphs showing the fractional Bo oriented parallel to the MRI image slices. hypointensity (1-S/So) as a function of dipole moment for 0080 FIGS. 47A-47C are theoretical magnetic resonance various isotropic (cubic) resolutions and echo times. images of single contrastagent particles comparing the signal 0100 FIG. 50A is a graph showing the fractional intensities of the particles at different positions within the hypointensity (1-S/So) as a function of dipole moment for a cubic voxels. 50-um isotropic resolution and an echo time of 5-ms. I0081 FIG. 47A shows the effect of position within the 0101 FIG. 50B is a graph showing the fractional Voxel on the signal intensity predicted for iron contrast agent hypointensity (1-S/So) as a function of dipole moment for a particles. 100-um isotropic resolution and an echo time of 5-ms. 0102 FIG. 50C is a graph showing the fractional I0082 FIG. 47B shows the effect of position within the hypointensity (1-S/So) as a function of dipole moment for a Voxel on the signal intensity predicted for nickel contrast 50-um isotropic resolution and an echo time of 10-ms. agent particles. 0103 FIG. 50D is a graph showing the fractional I0083 FIG. 47C shows the effect of position within the hypointensity (1-S/So) as a function of dipole moment for a Voxel on the signal intensity predicted for iron oxide contrast 100-um isotropic resolution and an echo time of 10-ms. agent particles. 0104 FIG. 50E is a graph showing the fractional 0084 FIG. 47D shows the positions of the contrast agent hypointensity (1-S/So) as a function of dipole moment for a particles within the voxel boundaries simulated in FIGS. 50-um isotropic resolution and an echo time of 20-ms. 47A-47C. 01.05 FIG. 5OF is a graph showing the fractional I0085 FIGS. 48A-48F are gradient-echo MRI images of hypointensity (1-S/So) as a function of dipole moment for a chemically synthesized and mirofabricated magnetic reso 100-um isotropic resolution and an echo time of 20-ms. nance contrast agents. 0106 FIG. 51 is a gradient-echo MRI image of microfab I0086 FIG. 48A is a gradient-echo MRI image using ricated iron disks Suspended in agarose. 50-um isotropic resolution of a prior art MPIO contrast par 0107 FIG. 52 is a TEM image of microfabricated single ticle. disk contrast agents attached to the cell membrane of a bio 0087 FIG. 48B is a gradient-echo MRI image using logical cell. 50-um isotropic resolution of a microfabricated solid nickel (0.108 FIG. 53 is a TEM image of microfabricated single contrast particle. disk contrast agents incorporated within the cell membrane of 0088 FIG. 48C is a gradient-echo MRI image using a biological cell. 50-um isotropic resolution of a microfabricated solid iron contrast particle. DETAILED DESCRIPTION 0089 FIG. 48D is a gradient-echo MRI image using 100 0109 The inventors have designed a magnetic resonance um isotropic resolution of a prior art MPIO contrast particle. structure (MRS) with a cavity or reserved space that provides 0090 FIG. 48E is a gradient-echo MRI image using 100 contrast and the additional ability to frequency-shift the spec um isotropic resolution of a microfabricated Solid nickel con tral signature of the NMR-susceptible nuclei such as water trast particle. protons by a discrete and controllable characteristic fre 0091 FIG. 48F is a gradient-echo MRI image using 100 quency shift that is unique to each MRS design. The fre um isotropic resolution of a microfabricated Solid iron par quency-shifted spectral signature, which may be engineered ticle. by controlling the precise geometry of the MRS, may be used 0092 FIGS. 49A-49F are histograms of single-voxel sig in addition to contrast to provide for identifying individual nal intensities from theoretical and experimental magnetic MRS’s in magnetic resonance (MR) image data or in any resonance images of microfabricated contrast agents normal other nuclear magnetic resonance system data, and for distin ized to the background signal intensity. guishing different MRS types/geometries from one another within these data. The individual magnitudes of frequency 0093 FIG. 49A is a histogram of theoretical single voxel shifting resulting from individual MRS may be associated signal intensity for a microfabricated iron contrast agent nor with an individual color on a color map of the spectral signa malized to the background signal intensity. tures acquired from each location or from each MRS within 0094 FIG. 49B is a histogram of the experimentally-mea an MR image, greatly enhancing the informational content of Sured single Voxel signal intensity for a microfabricated iron MR images. Using the MRS as contrast agents in MR imag contrast agent normalized to the background signal intensity. ing, the resulting MR imaging data may provide a color map 0095 FIG. 49A is a histogram of theoretical single voxel of the spectral signature shifts, which provides additional signal intensity for a microfabricated nickel contrast agent information regarding the identities of individual MRS, in normalized to the background signal intensity. addition to the contrast signals produced by the MRS. US 2011/0014129 A1 Jan. 20, 2011 0110. In the reserved space or cavity of the MRS a sub of the position of an individual MRS within a voxel using both stantially, spatially uniform magnetic field is generated the absolute value of the contrast signal and the relative con whose strength is significantly different from that of the back trast intensities from surrounding voxels. Thus, all MRS ground field outside the particle. The reserved space or cavity designs may be used as conventional T. magnetic resonance allows NMR-susceptible nuclei such as water protons to dif contrast agents with significantly improved efficacy relative fuse or flow in and out of the reserved space thereby increas to other T, contrast agents. The substantial uniform dimen ing the volume of fluid frequency-shifted during the repeated sions of these compositions also allow use of minimal detect application of resonant electromagnetic pulses. This diffu able concentrations in comparison to the Solid magnetic reso sion modulates the signal from a volume of fluid many times nance contrast agents required in the prior art. greater than the volume contained in the MRS, and this 0114. The ability to microfabricate both an MRS with a enhancement allows a lower-density of particles to be used in reserved space and a solid MRS from a variety of different, order to produce the contrast, in addition to the color infor and highly magnetic materials provides great advantages mation. because many of the paramagnetic materials currently used 0111. This frequency-shifting signal is produced by the for MRI contrast agents (for example, Gadolinium com MRS only if the MRS is exposed to a electromagnetic pulse at plexes) are considered potentially toxic at Some threshold a specific resonant frequency that is precisely specified by the amount. The MRS may be used in a number of applications particular design of the MRS. If the same MRS is exposed to including magnetic resonance frequency shifts of water pro a RF pulse with a frequency that is significantly different from tons and other NMR-susceptible nuclei for magnetic reso the resonance frequency of the MRS, no signal will be pro nance calibration/testing/fabrication, magnetic resonance duced. Individual MRS within an ensemble of MRS in a spatial calibration markers, specific detection/labeling/track sample, each having a different resonance frequency, may ing of biological cells, distance/pressure/vibration/torque produce a frequency-shifting signal when exposed to an RF sensors, torque/orientational measurements, magnetic sepa pulse at its characteristic resonance frequency with no signal ration, fluid pumps or mixers, localized RF magnetic heating production by the other MRS having different resonant fre elements, localized magnetic field gradients, microfluidic quencies in the ensemble. applications, flow cytometry, flow sensors for stents, and 0112 A group of essentially identical MRS particles hav single cell characterization. ing a reserved space and being essentially uniform in size and 0.115. A detailed description of embodiments of the MRS, composition may thus shift the frequency spectra of NMR methods of producing an MRS, and methods of using the Susceptible nuclei by the same discrete and controllable MRS is provided below. amount during exposure to resonant electromagnetic pulse. Different groups of MRS particles constructed to shift the 1. Definitions frequency spectra of NMR-susceptible nuclei by different 0116. The terminology used herein is for the purpose of discrete and controllable amounts may be used to perform describing particular embodiments only and is not intended to multiplexed magnetic resonance scanning in which the dif be limiting. As used in the specification and the appended ferent frequency spectrum shifts of the different MRS par claims, the singular forms “a,” “an and “the include plural ticles may be encoded as different colors in the resulting referents unless the context clearly dictates otherwise. magnetic resonance image. The MRS may be produced using 0117 For recitation of numeric ranges herein, each inter a technique that results in an essentially uniform size and vening number there between with the same degree of preci composition of a plurality of MRS. The combination of cre sion is explicitly contemplated. For example, for the range of ating a reserved space with an essentially uniform magnetic 6-9, the numbers 7 and 8 are contemplated in addition to 6 and field and a substantially pure composition both in material, 9, and for the range 6.0–7.0, the numbers 6.0, 6.1, 6.2, 6.3, 6.4. shape, and size, allow use of a relatively low detectable con 6.5, 6.6, 6.7, 6.8, 6.9, and 7.0 are explicitly contemplated. centration of a magnetic resonance contrast agent in a mag 0118 a. B. Magnetic Field netic resonance scan as compared to the amount of the MRI 0119. As used herein, a B magnetic field may be a uni contrast agent required in the prior art. form external applied magnetic field that possesses a uniform 0113. In addition to MRS structures that provide data that magnitude and uniform direction in the absence of any MRS may be used for color mapping, the inventors have also cre or other magnetic particles. The Bomagnetic field may also be ated nearly uniform solid magnetic resonance T contrast referred to as a background magnetic field. In certain appli agents that have a significantly higher magnetic moment cations, the Bomagnetic field may be produced by a magnetic compared to similarly-sized existing MRI contrast agents. resonance visualization device Such as an MRI scanner. Top-down fabrication method may be used to produce these b. Far-Field Volume solid MRS contrast agents from virtually any material, 0.120. As used herein, the far-field volume may be the including materials with a high Saturation magnetic density region outside the near-field volume of a MRS that encom Such as nickel or soft iron. As a result, the external magnetic passes the far-field magnetic field induced by the MRS struc fields produced by the solid MRS particles are significantly ture. stronger than the corresponding fields produced by existing c. Near-Field Volume MRI contrast agents such as Superparamagnetic iron oxide I0121. As used herein, the near-field volume may be a nanoparticles (SPIO). In fact, these solid magnetic resonance volume that is essentially centered on a MRS and extends out contrast agents increase visibility several-fold extending its from the structure to a distance of no more than a few times applications to areas such as in vivo single-cell tracking stud the maximum spatial dimension of the MRS itself. The extent ies. In addition, both MRS with a cavity/reserved space and of this near-field volume may scale with the size of the MRS. the solid particulate MRS, are dimensionally consistent from 0.122 d. Non-Magnetic Material particle to particle facilitating more quantitative image analy 0123. As used herein, a nonmagnetic material may be a sis and making possible Super-resolution tracking or locating material that does not exhibit a Substantial magnetic field US 2011/0014129 A1 Jan. 20, 2011 either intrinsically or when placed in a magnetizing field. I0131. In addition, the magnetic moment of the solid par Although nonmagnetic materials are distinguished from fer ticular MRS may be about 10'. Am, 10 Am, 10 Am, romagnetic materials and Superparamagnetic materials, non 10° Am, 10' Am, or 10'Am. Further, the variation in magnetic materials may not necessarily be completely non magnetic moment within a group having a particular speci magnetic in nature, but may include materials that are weakly fied magnetic moment may be within about 0.1%, about magnetic, very weakly paramagnetic or diamagnetic in 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, nature. For example, the water that is commonly detected and 9.0%, or 10% of the mean magnetic moment. The J. values of imaged in magnetic resonance systems is detected because of the solid particulate MRS may be 0.0T, 0.1T, 0.2T, 0.3T, 0.4 the nuclear magnetic resonance of the water. Because the T, 0.5T, 0.6T, 0.7T, 0.8T, 0.9 T, 1.0 T, 1.1 T, 1.2T, 1.3T, 1.4 magnetism of the water is extremely weak relative to the T. 1.5 T, 1.6 T, 1.7 T, 1.8 T, 1.9 T 2.0T, 2.1 T, 2.2T, 2.3T, 2.4 magnetic materials described herein, however, water and the T, or 2.5 T. other weakly magnetic materials described herein may be I0132) If the solid particulate MRS is a solid disk, the regarded as nonmagnetic materials. overall diameter of the solid disk MRS may range from about 0.5um to about 20 um, or about 0.5um, 1.0 m, 2.0 um, 3.0 2. Magnetic Resonance Structures um, 4.0 Lim, 5.0 um, 6.0Lm, 7.0 Lim, 8.0Lum, 9.0 um, 10.0 um, 11.0 um, 12.0 um, 13.0 um, 14.0 m, 15.0 lum, 16.0 um, 17.0 0.124 Provided herein is a magnetic resonance structure um, 18.0 um, 19.0 Lum, or 20.0 m. The overall thickness of (MRS). the solid disk MRS may range from about 0.5um to about 20 0.125 a. Solid High Magnetic Moment T. Contrast um, or about 0.5um, 1.0 um, 2.0 um, 3.0 Lim, 4.0 Lim, 5.0 um, Agents (Solid Particulate MRS) 6.0 m, 7.0 um, 8.0 Lim, 9.0 um, 10.0 um, 11.0 Lum, 12.0 um, 0126 The MRS may be a solid particle. The solid particu 13.0 um, 14.0 um, 15.0 um, 16.0 m, 17.0 m, 18.0 um, 19.0 late MRS may be high magnetic moment particles for high um, or 20.0 Lum. resolution imaging in which individual particles may be I0133. The solid particulate MRS may be composed of a located and tracked with greater precision for quantitative non-magnetic layer and/or a magnetic layer or combinations analysis. The solid particular MRS may share uniformity thereof. The thickness of each layer may vary between 1-nm from one particle to the next. The solid particular MRS may to 1000-nm in thickness or 1-mm, 10-nm, 20-mm, 30-nm, have a high magnetic moment because each particle uses 40-mm, 50-mm, 60-nm, 70-nm, 80-mm, 90-mm, 100-nm, 150 Substantially pure, strongly magnetic material. The Solid par nm, 200-nm, 250-mm, 300-nm, 350-nm, 400-nm, 450-nm, ticular MRS has these characteristics because it is generated 500-nm, 550-nm, 600-nm, 650-nm, 700-nm, 750-nm, 800 through top-down fabrication as discussed below. nm, 850-nm, 900-nm, 950-nm, 1000-nm, 1 um, 2 um, 3 um, 0127. The solid particulate MRS also may share unifor 4 um, 5um, 6 Jum, 7 um, 8 um, 9 um, 10um, 11 Jum, 12 um, mity in shape from one particle to the next. The minimum 13 Jum, 14 um, 15um, 16 Jum, 17 um, 18 um, 19um, or 20 Jum. detectable concentration of the solid particulate MRS when The magnetic material of the solid particulate MRS may be used as a magnetic resonance agent may be as low as an iron, nickel, chromium, manganese, cobalt, or any magnetic individual solid particulate MRS. The solid particulate MRS alloy Such as permalloy, neodymium alloy, alnico, bismanol, may be in the form of a disk, a cylinder, a pyramid, a cube, a cunife, fernico, heusler alloy, mkm steel, metglas, Samarium sphere, a rectangular block, a rod, a square, a crescent or any cobalt, sendust, or Supermalloy. The non-magnetic materials shape permutation thereof. The solid particulate MRS may that may be used as coatings or to provide cohesion between have a uniform shape and Surface or may have an uneven layers of the magnetic materials may be gold, titanium, Zinc, surface with protractions from the layer. silver, tin, aluminum, or any other material that does not 0128. The solid particulate MRS may be a T contrast generate a magnetic field. The Substrate layer used to generate agent. The high magnetic moment of the Solid particulate the Solid particulate MRS may be silicon, glass, quartz, Sap MRS may result in a stronger transverse dephasing of the phire, amorphous silicon dioxide, borosilicate or any other water protons around the particle, thus inducing a signifi inert Substance. The photoresistant material used to generate cantly higher T. contrast relative to existing magnetic par the solid particulate MRS may be positive/negative photore ticle T contrast agents such as MPIOs of similar size. sistant material Such as a polymethylmethacrylate, polymeth 0129. Further, the solid particulate MRS may have a very ylglutarimide, polymers, epoxy-based compounds such as low variability in the size of the particles or the composition SU-8, and phenol formaldehyde resins such as a mixture of of the material making up the particle. The size of each diazonaphthoduinone (DNO) and novolac resin. individual particles may vary by less than 10%, less than 9%, I0134. The solid particulate MRS may be in any form and less than 8%, less than 7%, less than 6%, less than 5%, less any consistency of the various Substrates, photoresistant than 4%, less than 3%, less than 2%, and less than 1% of the materials, and magnetic materials due to the photolitho mean size of the particles. As a result, the Solid particulate graphic patterning microfabrication techniques discussed MRS may be used in an advanced magnetic resonance visu below that allow arrays of many millions of solid particulate alization technique Such as Super-resolution tracking to a MRS that can be simultaneously fabricated. An exemplary much greater precision for quantitative analysis. solid particulate MRS is shown in FIG. 40. The solid particu 0130. The overall volume of a solid particulate MRS may late MRS has a 10-nm thick titanium adhesion layer that was range from about 5x10’m to about 5x10'm, or 5x10’ evaporated onto a Supporting Substrate made be silicon, glass, m,5x102 m, 5x102m,5x10'm,5x10 m, 5x10 quartz, Sapphire, amorphous silicon dioxide, borosilicate or 17 m, 5x10'm, or 5x10'm. The overall volume of each any other inert substance. A 100-nm layer of copper is laid Solid particulate MRS of a group having a particular specified over the titanium layer. Blocks of 300-nm thick layer of iron size may be consistently the same within about 0.1%, about or nickel surrounded by a 100-nm gold layer are laid across 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 6.0%, 7.0%, 8.0%, the copper layer through a bi-layer lift off process described 9.0%, or 10% of the mean volume below. Back-sputtered gold ion-milled from the substrate US 2011/0014129 A1 Jan. 20, 2011 redeposits on the iron/nickel sidewalls encase the entire solid 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2,0.3, particulate MRS of FIG. 41 leaving 100-nm thick top and 0.4,0.5,0.6, 0.7, 0.8, 0.9, 1.0, or 2.0. bottom gold coatings and 50-nm thick gold coatings along the 0.143 (1) Essentially Uniform Magnetic Field circumferential sidewall of the MRS particle of FIG. 41. FIG. 0144. The magnetic material of the MRS with a reserved 42 is a series of SEM images showing the resulting Solid space may produce a magnetic field throughout the near-field particulate MRS. Volume and far-field region. An essentially uniform magnetic 0135) b. MRS Contrast Agent with a Reserved Space field may be produced in the reserved space inside the near 0136. The MRS may comprise a reserved space. The field Volume, and a spatially decaying magnetic field may be reserved space may be situated within the interior of the produced in the volume external to the MRS. magnetic material or magnetic portions of the MRSSo as to be 0145 The sharpness and signal strength of the frequency at least partially surrounded by the magnetic material or mag shifting signal produced by the MRS depends most directly netic portions. The near-field Volume may comprise the on the characteristics of the essentially uniform magnetic reserved space. The size of the reserved space may be depen field within the reserved space of the MRS. In order to induce dent on the overall size and arrangement of the magnetic a detectably distinct characteristic Larmor frequency in any materials in the MRS. The reserved space may be in the form NMR-Susceptible material. Such as water protons passing of a disk shape, a tubular shape, a spherical shape, or any other through the essentially uniform magnetic field, the magnitude geometrical volume so long as the magnetic field formed of the essentially uniform magnetic field must be sufficiently within the reserved space is an essentially uniform magnetic different from the Surrounding magnetizing field. The mag field. The MRS may form at least one opening that permits nitude of the essentially uniform magnetic field may be speci fluid in the near-field volume to enter and exit the reserved fied by the selection of magnetic materials and arrangement space by diffusion, convection, or directional flow. The of the magnetic materials in the MRS. reserved space may be the main region in which the fre 0146 The magnetic material of the MRS may be selected quency-shifting of water protons and other NMR susceptible to have a particular Saturated magnetic polarization (J). nuclei occurs. The reserved space may be filled by a non resulting in an essentially uniform magnetic field within the magnetic fluid. reserved space (MRS field--background field) that is different 0.137 The magnitude of the frequency shift may be pre in magnitude from the background field magnitude, particu cisely controlled through variations in the magnetic strength larly when the magnetic material is magnetically fully satu of magnetic materials used to construct the MRS as well as rated. However, even when the magnetic material is only the relative proportions of the dimensions of the MRS. In partially magnetized, the essentially uniform magnetic field general, the magnitude of the frequency shift Act) may be may be sufficiently different from the background field if the expressed as: magnetic material has a sufficiently high J. Typically, the magnetic materials of the MRS will reach fully saturated I0138 where Y is the gyromagnetic ratio, J is the saturation magnetization within typical background MR fields. The magnetic polarization, and G is a dimensionless ratio of at detectable Larmor frequencies induced by the MRS may be least two linear dimensions that define the geometry of the relatively insensitive to the magnitude of the applied mag MRS. The gyromagnetic ratio and Saturation magnetic polar netic field of different magnetic resonance devices if the ization depend on the NMR-susceptible nuclei to be fre magnetic moment or moments of the MRS are fully saturated quency-shifted and the choice of magnetic material in the by background MR fields. MRS, respectively. The linear dimensions that define the 0147 Alternatively, if the magnetic material selected for geometry of the MRS are specified by the particular MRS the MRS is a permanent magnetic material Such as magnetite, structure and may include dimensions such as length, diam the essentially uniform magnetic field within the reserved eter, wall thickness, and others. The particular combination of space may be significantly different from the background dimensions that makeup G vary between MRS with different magnetic field even at relatively low (or Zero) background geometries. For example, for a dual-disc MRS, described in magnetic field magnitudes because the MRS generates a detail below: magnetic field independently of the background magnetic field. 0.148. The magnetic field may be a local region of interest 0139 where h is the disk thickness, R is the disk radius, within the near-field region of the MRS. This region may be and 2 S is the center-to-center disk separation. where the total magnetic field is substantially uniform and 0140. If the MRS structure is a hollow cylinder, also Substantially different in magnitude from any background described in detail below: magnetic field. The region of interest in which the essentially uniform magnetic field is induced by the MRS may not be G=LI(L’+(2p-i-ty’) '-(L’+(2p-t)’) ' (0.2) confined to be within the reserved space extending in a region 0141 where t is the cylinder wall thickness, 2p is the outside of the reserved space, but within the near-field region. cylinder diameter, and L is the length of the cylinder. Alternatively, the essentially uniform magnetic field may not 0142 For MRS with other structural geometries, similar extend through the entire reserved space. dimensionless ratios G may be derived using magnetostatic 014.9 The material to which the essentially uniform mag theory. However, because G is a dimensionless ratio of at least netic field induces a characteristic Larmor frequency may be two or more dimensions, the value of G is independent of the any material containing nuclei known in the art to be suscep overall size of the MRS structure. Depending on the particu tible to nuclear magnetic resonance due to the nuclei contain lar dimensions and structure of the MRS, G may vary between ing an odd number of protons or neutrons. Non-limiting about 0.1 and about 2. In various embodiments, G may be examples of nuclei susceptible to NMR include 'H, °fH, H, about 0.0001, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 13C, 10B, 11B, 11N, 15N, 17O, 19F, 2.Na, 29Si. 3 IP 35C1, 113Cd, 0.005, 0.01, 0.02, 0.03.0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 'Xe, and 'Pt. The material to which the essentially uni US 2011/0014129 A1 Jan. 20, 2011 form magnetic field induces a characteristic Larmor fre tained within a reserved space within the near-field region of quency may be water containing "H protons. the MRS during exposure to a resonant electromagnetic 0150 (2) Principle of Operation pulse. For example, the reserved space may be within a mag 0151. The MRS includes a near-field region in which an netizable shell or between neighboring magnetizable ele essentially uniform magnetic field significantly alters the ments. Within the reserved space, the MRS may function as a resonant Larmor frequency of the water protons or other specialized local magnetic field shifter. NMR-susceptible nuceli within the near-field region during 0158. The MRS may consist of specially shaped magne exposure of the MRS to a resonant electromagnetic pulse. tizable elements, which are exemplified by 102 and 194 in Magnetic resonance contrast is achieved by measuring the FIG. 1. Once magnetized to saturation by the background frequency shift of the near-field water protons and other magnetic field Bo (typically at least a few Tesla in magnitude), NMR-susceptible nuclei affected by the MRS magnetic field the specially shaped magnetizable elements may generate during the resonant pulse. localized regions of spatially homogeneous magnetic fields 0152. In general, magnetic resonance visualization tech within a reserved space, which are exemplified by 110 in FIG. niques are based on processing an electromagnetic signal 1. The spatially homogeneous magnetic fields may have a originating from water protons or other NMR-susceptible magnitude Substantially different from that of any Surround nuclei exposed to an applied magnetic field. In general, the ing magnetic fields. Hydrogen protons in the water molecules Larmor precession frequency () of a proton is induced to a or other NMR-susceptible nuclei present in these localized value that is directly proportional to a local magnetic field homogeneous magnetic field regions may experience a shift magnitude B according, as given in Eqn. (1): in Larmor precession frequency when the MRS is exposed to a resonant electromagnetic pulse, and the presence of the ()--YBo (1) magnetic resonance structure may be inferred via detection of 0153 where Y is the gyromagnetic ratio. The local mag these frequency-shifted NMR spectra. Signals originating netic field is typically dominated by the magnetic field from one particular type of the MRS may be differentiated applied by the magnetic resonance scanning device. Many from other types of MRS by using a type of MRS that induces existing MRI contrast agents, however, make use of magnetic discrete and controllable Larmor precession frequencies dur particles such as MPIOs to locally distort the applied mag ing resonant electromagnetic pulse that are detectably differ netic field of the magnetic resonance device to enhance the ent from the Larmor precession frequencies induced by the contrast of the resulting image in a local region Surrounding background magnetic resonance magnetic field and the local the contrast agent. magnetic field of the other types of MRS during electromag 0154) A magnetic object induces a magnetic field that netic pulses at their respective resonant frequencies. continuously decays in magnitude as a function of the dis 0159. The spatial profile and homogeneity of the local tance from the magnetic object within a relatively extended magnetic field within the reserved space may be accurately far-field Volume Surrounding the magnetic object. In the specified and controlled by the selection of magnetic materi vicinity of any magnetic structure, proton precession frequen als and the size, shape and arrangement of the magnetic cies vary proportionally to the spatially varying magnetic materials of the MRS. The degree of homogeneity of the local fields produced by that structure. Accordingly, NMR spectra magnetic field of the MRS directly influences the sharp defi integrating over NMR-Susceptible proton signals from nition of the resulting shifted nuclear magnetic resonance around that structure would typically integrate over broad (NMR) peaks. The spatial extent of the homogeneous mag frequency ranges, leading to broadened NMR spectral peaks. netic field directly influences the magnitude of the resulting 0155 Existing MRI contrast agents that include magnetic shifted color nuclear magnetic resonance (NMR) peaks. particles such as microparticles of iron oxide (MPIOs) gen Although the spatial extent of the homogeneous magnetic erate magnetic resonance contrast by locally altering the lon field is proportional to the physical sizes of the magnetizable gitudinal (T) or transverse (T or T) relaxation rates using elements of the MRS, the same is not true for the amount of these far-field effects. Because the far-field effects of these water protons and other NMR-susceptible nuclei that may existing MRI contrast agents involve non-homogeneous contribute to the frequency-shifted signal, due to the addi magnetic fields, however, no consistent and well-defined, tional effect of diffusion. quantized, and discrete color shift in the Larmor frequency of (0160 (3) Effect of Diffusion the water protons and other NMR-susceptible nuclei within 0.161 The diffusion of fluid into and out of the reserved the far-field region may be obtained using these existing MRI space within the near-field region effectively increases the contrast agents in the prior art. volume of frequency-shifted water protons or other NMR 0156 To yield instead a distinct frequency-shifted color susceptible nuclei by increasing the overall number of water NMR peak, the magnetic structure geometry of the MRS may protons or other NMR-susceptible nuclei influenced by the be such that it produces a fluid-accessible, extended spatial magnetic field within the reserved space. The diffusion effect volume over which the combined magnetic field from the significantly increases the contrast signal strength produced field of the MRS, together with the applied magnetizing back by MRS relative to a similarly-sized volume of fluid. ground magnetic resonance field Bo, is homogeneous and 0162. In the MRS, the number of water protons or other distinct in magnitude from the Surrounding magnetic fields. NMR nuclei that are exposed to the homogeneous field By contrast, the various embodiments of the MRS function as regions within each reserved space is enhanced by the con multispectral contrast agents by shifting the resonant Larmor tinual random self-diffusion of fluid containing NMR-sus precession frequencies of the water protons and other NMR ceptible nuclei in and out of each reserved space. The Susceptible nuclei in a discrete and controllable manner when enhanced magnitude of the shifted nuclear magnetic reso the MRS is exposed to a resonant electromagnetic pulse. nance (NMR) peaks due to these diffusion effects may benefit (O157. The MRS described herein may shift the NMR spec the MRS regardless of size, and may especially benefit from tra of NMR-susceptible nuclei such as water protons con a micrometer or smaller sized MRS. US 2011/0014129 A1 Jan. 20, 2011 0163. In the absence of diffusion effects, the effective time nucleus by about -10Hz, about -50 Hz, about -100 Hz, about for the replacement of frequency-shifted water protons and -150 Hz, about -200 Hz, about -400 Hz, about -600 Hz, other NMR-susceptible nuclei within the near-field region of about-800 Hz, about -1 kHz, about -10kHz, about -20kHz, a magnetic contrast particle is limited to a length of time on about -50 kHz, about -100kHz, about -200kHz, about -400 the order of the longitudinal relaxation time, T (2-3 sec.). The kHz, about-600kHz, about -800 kHz, about -1 MHz, about refresh time (t) for self-diffusion to refresh the fluid within a -2 MHZ, about -5 MHz, and about -10 MHz. reserved space of a magnetic resonance structure scales with the square of the structure's external dimension (R). As the 0169 Magnetic resonance imaging devices and methods size of the MRS is reduced, the saturated magnetization of may be used to obtain multispectral colormetric NMR fre NMR-susceptible nucleifalls only linearly with R, rather than quency-shift mapping using either direct imaging methods or in proportion to the structure's volume (R). Using the diffu indirect imaging methods. Using a direct imaging method, a sivity of water (2.3x10ms), the distance diffused during single excitatory electromagnetic pulse at the resonance fre the time T (6D-T)') is about 0.2 mm. Therefore, if the quency of each MRS is used to frequency-shift the NMR MRS is smaller than about 0.2 mm, the diffusivity effect susceptible nuclei within the reserved volume, followed by enhances the magnitude of the Saturated magnetization of NMR visualization. In this method, diffusion effects do not NMR-susceptible nuclei. enhance the strength of the NMR signal contrast because the 0164. Although diffusion is one mechanism by which MRS frequency-shift the NMR susceptible nuclei only dur water or other NMR-susceptible nuclei may move in and out ing the brief time of the excitatory electromagnetic pulse. of the reserved space resulting in enhancement of the fre Although the spatial resolution obtained using direct imaging quency-shift signal, NMR-Susceptible nuclei may move in is higher due to the concentration of frequency-shifted nuclei and out of the reserved space due to other mechanisms includ to the reserved space, the signal-to-noise ratio is relatively ing convection due to the flow of fluid in and out of the low. reserved space. The specific mechanism by which NMR 0170 Indirect imaging methods use a series oftemporally susceptible nuclei are transported in and out of the reserved separated excitatory electromagnetic pulses at the resonance space may depend on the specific structure of the MRS, the frequency of each MRS followed by NMR visualization of specific environment in which the MRS is to be used, and the the frequency-shifted nuclei. A significantly larger Volume of specific use of the MRS. NMR-susceptible nucleisuch as waterprotons are frequency 0.165 (4) Colormetric Frequency-Shifting shifted using this methods since fluid has sufficient time to (0166 The MRS may be designed to frequency-shift water diffuse in and out of the reserved space between excitatory protons or other NMR-susceptible nuclei by a wide range of pulses, effectively replenishing the reserved space with non discrete and controllable amounts relative to the background frequency shifted nuclei. As a result, the magnitude of the frequency-shift of surrounding NMR-susceptible nuclei. contrast signal is significantly increased, although the reso This frequency-shift signal of each MRS design may be used lution of the signal location is somewhat degraded due to the to identify each MRS individually within magnetic resonance diffusion of the frequency-shifted nuclei throughout the near imaging data. At least two MRS may be designed to fre field region of the MRS and beyond during the series of quency-shift the NMR-susceptible nuclei by discrete and excitatory pulses. controlled amounts such that the frequency-shift of each (0171 (5) Minimum Detectable Concentration MRS is distinguishable from the background frequency-shift 0172. In order for an MRS to be detected, the contrast as well as the frequency-shift of any of the other MRS. The signal must exceed the background noise. In the case of T individual magnitudes of NMR frequency-shifting resulting contrast signaling, the contrast signal may result from inter from individual MRS may be associated with an individual actions of NMR susceptible nuclei with the rapidly decaying color on a color map of the spectral signatures of the indi magnetic field external to the MRS. In this case, the strength vidual Voxels within an MR image, greatly enhancing the of the contrast signal may be governed by the magnitude of informational content of MR image data. This effective color the magnetic moment produced by the MRS within the back signal provides additional information regarding the particu ground magnetic field. In order to be detected using typical lar configuration of the MRS in the nuclear resonance image. researched level high-resolution magnetic resonance visual 0167 As described in detail elsewhere in this application, ization methods, the minimum magnetic moment may be the frequency-shift induced by a particular MRS may be about 10 Am, 10 Am, 10 Am, 10° Am2, controllably and consistently specified by a combination of 10' Am, or 10'A'm. This exact required minimum will the magnetic material included in the MRS and the shape, depend on imaging resolution and background noise levels dimensions, and separation distances of the magnetic struc particular to imaging protocols and imaging equipment. For tures included in the MRS. Using a top-down fabrication low resolution imaging that may include routine clinical low process, described in detail below, to produce the MRS, mag resolution imaging the minimum magnetic moment may be netic materials having a wide range of magnetic properties higher than all these numbers. may be formed into highly reproducible MRS configurations 0173 The magnetic moment typically depends on the vol with precisely defined reserved spaces. As a result, the MRS ume of magnetic material in the MRS as well as the saturated particles may be designed and produced to reliably fre magnetic polarization J of the magnetic material, a measure quency-shift NMR-susceptible nuclei by an amount that is up of the magnetic strength of the material. As a result, an MRS to several orders of magnitude higher than any existing constructed from a material with a high J. Such as iron, may chemical-shift MRI contrast agent. produce a detectable magnetic moment using a much smaller 0168 The MRS may be designed and produced to fre Volume of magnetic material compared to existing magnetic quency-shift a NMR-susceptible nucleus by any amount particle contrast agents, such as MPIOS. For example, if the ranging from about -10 Hz, up to about -10 MHz. Other MRS is a solid disk made of iron (J-2.2), a single particle designs of the MRS may frequency-shift a NMR-susceptible having a diameter of about 0.5-lum may produce a Suitably US 2011/0014129 A1 Jan. 20, 2011 high magnetic moment for detection using typical high reso size decreases. Thanks to diffusion, the required concentra lution NMR visualization methods. tion reduces quadratically with the MRS size. 0.174 For MRS having a reserved space, and being used 0177. The minimum detectable concentration of the MRS not in the T contrast mode, but in their multispectral fre may be well below that of existing contrast agents such as quency shifting mode (described above), the contrast signal chemical exchange contrast agents, gadolinium relaxivity strength may result from the interactions of NMR-susceptible based contrast agents and may be comparable to the mini mum detectible concentration of existing SPIO contrast nuclei Such as water protons within the reserved space during agents. Further, since existing gadolinium and SPIO agents the time that the MRS is exposed to an excitatory electromag are not spread evenly throughout the body after administra netic pulse at the resonance frequency of the MRS. If the tion, the minimum detectable concentration of the MRS may MRS is exposed to multiple excitatory pulses, diffusion be far below that of the actual detected concentrations of other effects enhance the volume of frequency-shifted nuclei as exiting agents including SPIO contrast agents. described above, resulting in a stronger contrast signal com 0.178 The minimum detectible concentration may also be pared to a similarly-sized MRS lacking the diffusion effects. quantified as the minimum number of contrast particles per Consequently, the strength of the signal, which is propor unit volume that may be detected by typical magnetic reso tional to the volume of frequency-shifted nuclei, may be nance visualization devices. For all MRS, including solid many times greater than the Volume of the reserved space, particulate MRS and MRS including a reserved space, single thanks to the contribution of the natural diffusion effects. The particles may be detected using typical existing magnetic contrast signal must exceed the background signal levels in resonance visualization devices such as MRI scanners. As a order to be detected. result, the minimum number of particles that may be detected 0175 Because the contrast signal strength of a MRS with per unit volume may often be as low as one particle per unit a reserved space depends in part on diffusive effects, the Volume. The ability to detect single particles depends in part overall size of the MRS is a significant factor. Ultimately, the on the overall size of the particle, as discussed above for both minimum useable size of the MRS may not be limited by the solid particulate MRS and the MRS with a reserved space, fabrication techniques, but by the refresh time (t) for self in addition to the image resolution of the magnetic resonance diffusion. Ideally, fast diffusion helps increase the volume of visualization device. Further, in order to discriminate water contributing to contrast signal, but the diffusional between two or more individual particles the particles may exchange of fluid in and out of the reserved space of a mag need to have a minimum separation distance. For the case of netic resonance structure should not be so fast as to broaden solid MRS, this minimum would be at least one imaging the peak of the NMR signal by more than the shift of the NMR voxel. For the case of cavity/reserve space MRS, this mini peak relative to the NMR peak shift. Because the MRS is mum can be far Smaller that even a single Voxel because the capable of generating sizeable NMR peak frequency shifts, frequency discrimination can be used to separate the two. In the diffusional broadening of the NMR peak becomes signifi order to minimize, however, the signal distortion due to inter cantly limiting for structures below about 100 nm in size, ference of the magnetic field of one MRS with a second MRS, where the magnetic material concentrations required are in the MRS may be separated by a distance of at least 2-3 times the nanomolar regime. The magnitude of the NMR signal, the the overall size of the MRS, which will generally still be many shift of the NMR signal peak relative to water protons and times Smaller than an individual Voxel size. other NMR-susceptible nuclei in the far-field region, and the 0179 c. Geometric Arrangements of Magnetic Material width of the shift NMR signal peak are all dependent on the for Reserved Space MRS materials and geometry of the MRS. 0180. The magnetic resonance contrast provided by the 0176). If continual longitudinal relaxation is assumed, the reserved space MRS is highly sensitive to its size and arrange magnetic moment Saturated out of each magnetic resonance ment of the magnetic materials. The magnetic material may structure pulsed over a time t-2 T is (m/2)*(T/t)*(1- form the reserved space within a continuous structure, or e'). Because the signal-to-noise ratio (SNR) varies with the within an arrangement of two or more magnetic portions. The Voxel Volume of the magnetic resonance imaging device, at two or more magnetic portions may be separate structures, or least about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10% different portions of an integral structure. The two or more fractional saturation of the water protons or other NMR magnetic portions may beformed in any shape and held in any susceptible nuclei may be needed for reliable detection of an arrangement Such that an essentially uniform magnetic field MRS. The minimum detectable concentration of the MRS is formed within the reserved space when the MRS is placed with a reserved space may be about 10M, 10' M, 10' in a magnetizing field. The reserved space MRS may be any M, 10 M, 10° M, or 10' M, depending on the overall variance or defamation in shape or thickness of the MRS. The size and magnetic material of the NMR, and the resolution reserved space MRS may be a dual disk MRS. The reserved and background noise of the NMR imaging device. For space MRS may also have a tubular or hollow shape, such as example, if the MRS with reserved space is a about 1 a hollow cylinder, a spherical shell, a rod, an elliptical shell, a micrometer in overall size, the minimum detectable concen shell with multiple small holes, or any other hollow shell tration may be about 10 M. In general, smaller sized MRS shape. For example, the reserved space MRS may be a will have lower detectable concentrations than larger MRS, slightly curved cylinder or may be a disc that has varying due to the relatively higher contribution of diffusion effects in thickness over the contours of the disk. the Smaller structures. That is, although the required molar 0181. The magnetic material may form at least one or concentration (representing a measure of number of indi more openings to allow fluid to freely diffuse and/or flow in vidual MRS’s) must increase as MRS sizes decrease, the total and out of the reserved space inside the near-field volume. amount of material required (which is of course less for each The total Surface area occupied by the one or more openings smaller MRS) will go down overall, leading to a highly favor formed by the magnetic material may range from about 0.1% able scaling of required material concentrations and MRS to about 90% of the total outer Surface area of the MRS. The US 2011/0014129 A1 Jan. 20, 2011 one or more openings may also occupy a total Surface area 0186 Each spacer may be formed from a non-magnetic ranging from about 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, material. The non-magnetic material of the spacer may be an 0.7%, 0.8%, 0.9%, 1.0%, 1.5%, 2.0%, 2.5%, 3.0%, 3.5%, internal metal post, a photo-epoxy post, a biocompatible 4.0%, 4.5%, 5.0%, 5.5%, 6.0%, 6.5%, 7.0%, 7.5%, 8.0%, material, hydrogel, or various polymer materials. For 8.5%, 9.0%, 9.5%, 10% to about 20%, from about 15% to example, the nonmagnetic material of the spacers may about 25%, from about 20% to about 30%, from about 25% to expand or contract as a function of temperature. As tempera ture varies, the spacing between the two or more magnetic about 35%, from about 30% to about 40%, from about 35% to portions may increase or decrease, thereby changing the mag about 45%, from about 40% to about 50%, from about 45% to nitude of the essentially uniform magnetic field within the about 55%, from about 50% to about 60%, from about 55% to reserved space, resulting in a different spectral shift of water about 65%, from about 60% to about 70%, from about 65% to protons and other NMR-Susceptible nuclei during magnetic about 75%, from about 70% to about 80%, from about 75% to resonance probing. The materials of the spacers may decom about 85%, or from about 80% to about 90% of the total outer pose, or disconnect from the magnetic portions, thereby dis surface area of the reserved space MRS. The total outer sur rupting the arrangement of the magnetic portions and elimi face area of the reserved space MRS is dependent on its nating the internal uniform magnetic field inside the reserved overall size and shape. space entirely. The spacing of the disks may be altered in 0182. The MRS may comprise a reserved space enclosed response to various physiological and chemical factors by by a semipermeable material to allow fluid to move in and out changing the geometry of the spacer. In addition, the dual of the reserved space via diffusion or convection. The semi disk MRS may be simply inactivated by disintegration of the permeable material may be any biological or synthetic semi spacer element. permeable material known in the art. The semipermeable 0187. The magnetic material of the dual-disk MRS may be material may allow certain molecules or ions to pass through iron, nickel, a hybrid material, or mixtures thereof. The mag by diffusion or facilitate diffusion. The semipermeable mem netic disk may be layered with different magnetizable mate brane may be a phospholipid bilayer, a nanoporous polymer, rials such as iron, nickel, chromium, manganese, cobalt, or a microporous polymer, a cell membrane, a thin film com any magnetic alloy such as permalloy, neodymium alloy, posite membrane, polyimide, cellulose ester membrane, alnico, bismanol, cunife, fernico, heusler alloy, mkm steel, charge mosaic membrane, bipolar membrane, anion metglass, Samarium-cobalt, sendust, or Supermalloy. The exchange membrane, alkali anion exchange membrane, and magnetic disk may be coated with non-magnetic materials proton exchange membrane. The MRS may comprise a such as gold, titanium, zinc, silver, tin, aluminum, or any reserved space that is enclosed by a non-permeable material other material that does not generate a magnetic field. These Such as gold or titanium coatings thereby trapping the fluid non-magnetic materials may also be used to provide a cohe inside. For example, the MRS may a completely package sive layer between two other magnetic material layers of the reserved space for use in microfluidic applications as dis disk. Each of these layers may have a thickness of 1-10 nm, cussed below. 1-nm, 2-mm, 3-nm, 4-nm, 5-mm, 6-mm, 7-mm, 8-mm, 9-mm, 0183) (1) Dual-Disk Magnetic Resonance Structure 10-nm, 20-mm, 30-nm, 40-nm, 50-nm, 60-nm, 70-nm, 80-nm, 0184 The reserved space MRS may be in the form of a 90-nm, 100-nm, 150-mm, 200-nm, 250-nm,300-nm,350-nm, dual-disk magnetic resonance structure (MRS). The dual 400-nm, 450-nm, 500-nm, 600-nm, 700-nm, 800-nm, 900 disk MRS may include two disk-shaped magnetic portions nm, 1000-nm, 1-um, 2-lum, 3-um, 4-um, 5-lum, 6-um, 7-um, held apart at a fixed distance by one or more non-magnetic 8-um, 9-um, 10-um, 20-um, 30-um, 40-um, 50-um, 60-um, Support elements. The open geometry of the dual-disk design 70-um, 80-lum, 90-um, 100-um, 150-lum, 200-um, 250-lum, may enhance the accessibility of the reserved space to the 300-um, 350-um, 400-um, 450-um, 500-lum, 550-lum, 600 diffusive and/or convective exchange of fluid. The one or um, 650-um, 700-um, 750-um, 800-um, 850-um, 900-um, more non-magnetic Support elements may be in the form of 950-um, 1000-um, 1-mm, 2-mm, 3-mm, 4-mm, 5-mm, one or more spacers arranged between the two or more mag 6-mm, 7-mm, 8-mm, 9-mm, 1-cm, 2-cm, 3-cm, 4-cm, 5-cm, netic portions of the MRS, or one or more spacers arranged to 6-cm, 7-cm, 8-cm, 9-cm, or 10-cm. be located external to the reserved space between the two or 0188 The magnetic disks may be constructed from mate more magnetic portions. rials that are magnetized by a background magnetic reso 0185. The spacer arranged between two or more magnetic nance field that is much larger in magnitude than the essen portions may maintain the reserved space Such that the tially uniform magnetic field generated by the dual-disk reserved space is open to permit a fluid to flow in and out of MRS. Because of the quadrature vector addition of magnetic the reserved space or enclosed area of fluid. The spacer may fields, only those components of the essentially uniform mag be arranged to partially or completely fill the reserved space netic field that are parallel or antiparallel to the background between the magnetic portions to prevent the movement of magnetic resonance field need be substantially uniform and fluid in or out of the reserved space. The nonmagnetic mate homogeneous. The dual-disk MRS may also be constructed rial of the spacer may have different properties in different from a permanent magnetic material and may be used with environments, including Surrounding pH, temperature, and our without background magnetic field. The entire essentially Solution salinity. These environmentally-dependent proper uniform magnetic field of the reserved space may be substan ties of the nonmagnetic spacer material may be utilized to tially uniform and homogeneous. produce a change in the essentially uniform magnetic field or (0189 The disks of the double disk MRS may have a thick within the reserved space. These changes produce detectable ness (h) of 1-mm, 2-mm, 3-nm, 4-nm, 5-mm, 6-mm, 7-mm, changes in the signals produced by the dual-disk MRS during 8-mm, 9-mm, 10-nm, 20-mm, 30-nm, 40-nm, 50-nm, 60-nm, observation with a magnetic resonance system, or change to 70-nm, 80-mm, 90-mm, 100-nm, 150-mm, 200-nm, 250-nm, block or unblock the reserved space thereby making the 300-nm, 350-nm, 400-nm, 450-nm, 500-nm, 550-nm, 600 reserved space inaccessible or accessible to fluid. nm, 650-nm, 700-nm, 750-mm, 800-nm, 850-nm, 900-nm, US 2011/0014129 A1 Jan. 20, 2011 950-nm, 1000-nm, 1-um, 2-um, 3-um, 4-um, 5-lum, 6-um, a case in which the MRS 100 is not embedded in a magne 7-um, 8-um, 9-um, 10um. The radius (R) of the disc may be tizing field 108 while in use. A local region of interest may 2-mm, 5-mm, 6-mm, 7-mm, 8-mm, 9-mm, 10-nm, 15-mm, 20-nm, include the central portion of the region between the two 25-mm, 30-nm, 35-mm, 40-nm, 45-mm, 50-nm, 60-nm, 70-nm, spaced magnetic disks, such as the disks shown in FIG.1. The 80-mm, 90-mm, 100-nm, 150-nm, 200-nm, 250-mm, 300-nm, total magnetic field in the reserved space 106 may be a com 350-nm, 400-nm, 450-nm, 500-nm, 550-nm, 600-nm, 650 bination of the local magnetic field created by the MRS 100 nm, 700-nm, 750-nm, 800-nm, 850-mm, 900-nm, 950-nm, and a portion of a background magnetic field when the mag 1000-nm, 1-um, 2-um, 3-um, 4-um, 5-lum, 6-um, 7-um, 8-um, netic resonance structure 100 is embedded in the background 9-um, 10-um, 20-um, 30-um, 40-um, 50-um, 60-um, 70-um, field during use. Alternatively, the local region of interest may 80-lum, 90-um, 100-um, 150-lum, 200-um, 250-lum, 300-um, include the control portion of the region between the two 350-um, 400-um, 450-um, 500-lum, 550-lum, 600-um, 650 spaced magnetic disks Such as the disks shown in FIG. 1. um, 700-um, 750-um, 800-um, 850-um, 900-um, 950-lum, 0.192 FIG. 2 shows the calculated distribution of the mag 1000-um, 1-mm, 2-mm, 3-mm, 4-mm, 5-mm, 6-mm, 7-mm, nitude of the magnetic field corresponding to the MRS of 8-mm, 9-mm, 7-mm, 8-mm, 9-mm, 1-cm, 2-cm, 3-cm, 4-cm, FIG.1. The dual-disk MRS generates a highly homogeneous 5-cm, 6-cm, 7-cm, 8-cm, 9-cm, 10-cm, 20-cm or 30-cm. The magnetic field over a large Volume fraction, as shown in FIG. center to center separation (2S) between the dual disks may be 2, and the open design helps maximize fluid self-diffusion 50-nm, 60-nm, 70-nm, 80-mm, 90-mm, 100-nm, 150-nm, 200 and/or convection that dramatically increases its signal-to nm, 250-mm, 300-nm, 350-nm, 400-nm, 450-nm, 500-nm, noise ratio (SNR) over that of existing closed structure MRI 550-nm, 600-nm, 650-nm, 700-nm, 750-nm, 800-nm, 850 contrast agents, as discussed above. In addition, the dual-disk nm, 900-nm, 950-nm, 1000-nm, 1-um, 2-lum, 3-um, 4-lum, MRS is inherently scalable and well-suited to massively par 5-um, 6-um, 7-um, 8-um, 9-um, 10-um, 20-um, 30-um, allel wafer-level microfabrication techniques explained in 40-um, 50-um, 60-um, 70-um, 80-lum, 90-um, 100-um, 150 detail below. The discs 102 and 104 may be held in position by um, 200-pm, 250-lum, 300-um, 350-um, 400-um, 450-lum, non-magnetic spacers that may include an internal metal post 500-lum, 550-lum, 600-um, 650-um, 700-um, 750-um, 800 (see FIG. 8) or one or more external biocompatible photo um, 850-um, 900-um, 950-um, 1000-um, 1-mm, 2-mm, epoxy posts (see FIG. 9). 3-mm, 4-mm, 5-mm, 6-mm, 7-mm, 8-mm, 9-mm, 10-mm, 0193 The Larmor frequency shift Act) relative to the Lar 20-mm, 30-mm, 40-mm, 50-mm, or 100-mm. The saturation mor frequency of the water protons and other NMR-suscep magnetic polarization (Js) may be 0, 0.1, 0.2,0.3T, 0.4T. 0.5 tible nuclei located in the far-field region of the MRS may be T, 0.6T, 0.7 T, 0.8 T, 0.9 T, 1.0 T, 1.5 T 2.0 T, or 2.5 T. approximated analytically using the estimated magnetic field (0190. The double disk MRS may be as shown in FIG.1. In strengthat the center of the dual-disk MRS. Using elementary this embodiment, the MRS 100 includes two magnetic disks magnetostatics analysis, the Larmor frequency shift Act) near 102 and 104—an upper magnetic portion 102 and a lower the center of the reserved area for the MRS including a pair of magnetic portion 104 that are arranged at a constant distance magnetically saturated disks may be determined using the from each other, forming a reserved space 106 between the relationship given in Eqn. (2): magnetic portions. The reserved space 106 may be filled with a non-magnetic material. Such as a fluid, which may be water, 2+R2)/2) (2) a paste, a gel or a gas. The non-magnetic material may flow 0194 where Y is the gyromagnetic ratio, h is the disk and/or diffuse through at least a portion of the reserved space thickness, R is the disk radius, 2S is the center-to-center disk 106. When the magnetic portions 102 and 104 are placed separation, and J is the saturation magnetic polarization. For within a magnetizing field 108, magnetic moments 114 and thin discs with h<2Ss.R., this reduces to Eqn. (3): 116 and associated magnetic fields 110 and 112 are induced. In the reserved space 106, the induced magnetic fields 110 and 112 interact to form an essentially uniform magnetic hR? (3) field. This essentially uniform magnetic field induces the Aco & -y, 22 so.2S2)32 nuclear magnetic moments of any material passing through the reserved space 106, such as water molecules or other NMR-susceptible nuclei, to precess at a characteristic Lar 0.195 The Larmor frequency shift may be specified by mor frequency if the NMR is exposed to a resonant electro modifying any of the quantities specified in Eqn. (3), includ magnetic pulse and if the combined magnetic field is uniform ing J, h, R. S. and combinations thereof. For example, if the (background+reserved space). The magnitude of the essen disks are constructed of soft iron, which has a J of about 2.2 tially uniform field in the reserved space is sufficiently dif Tesla, a Larmor frequency shift of about -10 MHZ may be ferent from all surrounding fields such that the characteristic achieved. Larmor frequency of the NMR-susceptible material passing 0196. The estimates of Larmor frequency shifting through the reserved space 106 during exposure of the MRS described above implicitly assume alignment between the to a resonant electromagnetic pulse and is detectably different disc planes and the applied magnetizing magnetic resonance from the Larmor frequency induced by the background field field, Bo. Such alignment may be passively maintained by the 108 in the absence of the MRS 100. The characteristic Larmor inherent magnetic shape anisotropy of the MRS, as shown in frequency is identifiable with the particular arrangement and FIG. 10. For any misalignment angle (0) between Bo and the choice of materials making up the MRS 100. disk planes, the resulting magnetic torques on the discs pro 0191 The total magnetic field in the reserved space may duce an automatic self-aligning pressure of approximately be equal to the local magnetic field (reserved field space and (h/(R+S)1/2)(Js/lo) sin(20), equating to a pressure of background field) created by the MRS. The total magnetic about 10 to about 10 N/um. By comparison, within cel field in the reserved space 106 may be equal to the combined lular cytoplasm, the yield stresses range from about 10' to local magnetic fields 110 and 112 created by the MRS 100, in about 10 N/um. US 2011/0014129 A1 Jan. 20, 2011 0197) The relatively high homogeneity of the essentially free to rotate about a central axis parallel to Bo. Because the uniform magnetic field (MRS field--background field) within resulting NMR color frequency shifts are invariant with the reserved space of the dual-disk MRS may suppress the respect to this rotation, a variety of alternative structures, each background magnetic resonance signal while still Saturating composed of what may be regarded as Superpositions of out about/3 of the volume between the discs via off-resonant rotated dual-disk structures, may also possess the appropriate magnetic resonance excitation pulses with bandwidths of just homogeneous field profiles. Although a hollow cylinder rep a few percent of the shift of the MRS, as shown in FIG. 6. For resents a surface of revolution of a radially-offset thin rect an equilibrium Bo-aligned magnetization, Mo, and h-2Ss.R., angle, rather than a disk shape, the similarity of the hollow the magnetic moment of the NMR-susceptible nuclei satu rated in a single excitation pulse is ma-MotR/3. Since cylinder structure MRS to a rotated dual-disk MRS means not all of the fluid within the near-field region exchanges that the magnetic fields in the reserved space may likewise between consecutive excitation pulses, however, this pre generate distinct spectrally shifted color NMR peaks. pulse magnetic Saturation Volume falls with Subsequent (0204. The hollow cylinder MRS may be scalable down to pulses. For an inter-pulse delay (t) of R/6D, simulations the nano-regime with an optimal length-to-diameter ratiojust indicate that a resulting per-pulse average saturation may be above unity. The hollow cylinder MRS is defined by the about m/2. The spatial distribution of any single excita overall Saturation magnetic polarization (Js), wall thickness tory pulse of Saturated magnetization at Some later time, (t), diameter (2p), and length (L). The wall thickness (t) of the tot, may be approximated by analogy to an instantaneous hollow cylinder MRS may be 1-nm, 2-mm, 3-nm, 4-nm, 5-nm, point-source diffusion problem, giving Eqn. (4): 6-mm, 7-mm, 8-mm, 9-mm, 10-nm, 15-mm, 20-mm, 25-mm, M.(r,t)-(me/2)(4J.Dt)' exp(-r/4Dt)exp(-t/T) (4) 30-nm, 35-mm, 40-nm, 45-mm, 50-nm, 55-mm, 60-nm, 65-mm, 70-nm, 75-mm, 80-mm, 85-mm, 90-mm, 95-mm, 100-nm, 110 0198 where the final factor accounts for relaxation back into alignment with Bo, and r measures the distance from the nm, 120-mm, 130-nm, 140-mm, 150-mm, 160-nm, 170-nm, MRS. Within a characteristic diffusion distance, d=(D;T)', 180-mm, 190-nm, 200-nm, 250-mm, 300-nm, 350-nm, 400 a t-spaced train of such excitatory pulses rapidly (overa time nm, 450 nm, and 500-nm, 550-nm, 600-nm, 650-nm, 700 of approximately T) asymptotes to a steady-state distribu nm, 750-nm, 800-nm, 850-nm, 900-nm, 950-nm, 1000-nn, tion given by Eqn. (5): 2-um, 3-um, 4-um. 5-lum, 10-um, 100-um, 200-um, 300-um, 400-um, 500-lum, 600-um, 700-um, 800-um, 900-um, or 1 mm. The diameter (2p) may be 50-nm, 100-nm, 150-nm, (0199. By integrating Eqn. (5) over a spherical voxel of 200-nm, 250-mm, 300-nm, 350-nm, 400-nm, 450-nm, 500 radius RdR with R to about 2T, and may have a J of 0, 0.1, 0.2,0.3, 0.4,0.5,0.6, and may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0 80, 90, 100, 100, 200, 300, 400, 500, 600, 700, 800, 900 or T. 2.1 T, 2.2T, 2.3T, 2.4T, or 2.5 T. For example, the magnetic 1000 nm, or 2, 3,4,5,6,7,8,9, 10, 20, 30, 40, 50, 60, 70, 80, material included in the MRS may be soft iron with a J 2.2 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 um, TThe magnetic material may also be nickel with a J ranging or 2, 3, 4, 5, 6, 7, 8, 9, or 10 mm, or 2, 3, 4, or 5 cm. The size from about 0.5 T to about 0.6T. The magnetic material may be of the MRS may be selected according to its particular appli iron oxide with a J-0.5 T. The magnetic material may also cation or use. In applications such as blood flow visualization have a magnetic moment that is saturated when the magnetic or perfusion imaging, the MRS may range in size from about 1 mm to about 5 cm. The size of the MRS may be matched material is placed in magnetizing fields having strength with the size scale of a particular blood vessel. For example, within the operating capacity of a magnetic resonance visu the upper size limit of the MRS may match the size of a human alization system. aorta. The MRS may also be larger for use in applications 0217. The magnetic material may be a single magnetic Such as industrial flow visualization. material, or a combination of two or more magnetic materials. The combination of two or more magnetic materials may be 0223) The MRS may also have a maximum dimension in the form of an alloy in which the magnetic materials are between about 10 nm and about 100 Lum. The MRS may be combined in a homogenous mixture, or in the form of a Smaller than about 10 nm to approach molecular size, and layered magnetic structure in which two or more magnetic may be particularly useful in micro-tagging applications. The materials are formed into two or more discrete layers. Each MRS may also have a maximum dimension ranging from layer may be made up of a different magnetic material thanan about 50 nm to about 10 um, which may facilitate cellular adjoining layer. uptake in a biological, diagnostic and/or medical application. 0218. e. Hybrid Materials and Nonmagnetic Materials 0224 g. Activatable MRS 0219. The non-magnetic materials, or hybrid materials 0225. The MRS may be used as a “smart” indicator by containing a mixture of one or more magnetic and non-mag disrupting the diffusion or flow of fluid into and out of the netic materials may be used to construct the MRS in addition reserved space using an additional external coating or by to the magnetic materials. These non-magnetic or hybrid filling in the reserved space with a material. By preventing the materials may be used to position the magnetic materials in a access of fluid into the reserved space, the frequency-shifting spatial arrangement Suitable for forming a reserved space, to function of the MRS is effectively inactivated. The external modify the diffusion and or flow of fluids in and out of the coatings or filler materials may be selected to disintegrate reserved space, to reinforce the strength of the magnetic under selected conditions in order to activate the frequency materials, and to impart desirable surface properties to the shifting function of the MRS. Once the external coatings or MRS such as biocompatibility, cell-specific affinity, or hydro filler have disintegrated, the frequency-shifting function of phobicity. Further, magnetic materials may be deliberately the MRS is irreversibly activated. mixed with non-magnetic materials in order to modify the 0226. The specific structure of the dual-disk MRS may be magnetic properties of the resulting mixture. The non-mag used as a “smart” indicator by selecting a material for the netic materials may include non-magnetic metals such as spacer between the disks that either disintegrates, Swells, copper, titanium, and gold. In addition, the non-magnetic and/or shrinks in response to changes in environmental fac materials may include non-metals such as a ceramic, a plastic, tors such as temperature, pressure, pH, salinity, presence of an or a photoresist material. The non-magnetic materials may be enzyme, and others. If the spacer material disintegrates, the physically separate from the magnetic materials, or the non dual disks are separated and the frequency-shifting function magnetic materials may be mixed or interspersed among the is irreversibly disabled. If the spacer material, which may be magnetic materials in the form of particles or layers to form a hydrogel, Swells or shrinks in response to an environmental the hybrid material. factor, the spacing between the disks is altered, resulting in an 0220. The hybrid material may include two or more alter altered magnitude of frequency shift, due to the dependence nating magnetic and non-magnetic layers, and/or a conglom of the magnitude of the frequency shift on the relative posi eration containing Smaller particles of magnetic material tioning and dimensions of the magnetic elements of the dual embedded within a host non-magnetic material. The hybrid disk MRS, including the disk spacing. Thus, a dual-disk MRS element may be a magnetic material and/or layered magnetic having a Swellable spacer material produces a frequency shift structure with an outer coating made of a non-magnetic mate that reversibly changes to reflect changes in an environmental rial. The non-magnetic coating may be an oxidation or cor factor. rosion barrier, a mechanical strengthening layer, a non-toxic 0227. A spectrally distinct physiological “smart” indica coating, a biologically inert coating such as titanium, or a tor may also be formed by either encapsulating the MRS, or coating to facilitate common bioconjugation protocols such filling its reserved spaces, to inhibit internal diffusion or flow as gold. The gold coating may be further functionalized using (as shown in FIGS. 17 and 23E), while leaving the far-field a technique Such as thiol-based chemistry. In addition, the spatially trackable image-dephasing capabilities unaffected. non-magnetic coating may include a coating applied to act as In the encapsulated MRS, the frequency-shifted signal would a non-magnetic buffer Zone to inhibit magnetic clumping of be limited to the signal produced by any NMR susceptible multiple MRS particles, to improve field uniformity by physi nuclei encapsulated within the reserved space of the MRS. If cally excluding access to select Surrounding spatial Volumes the diffusion-inhibiting or convection-inhibiting material is over which fields might be less uniform than desired, to vary chosen to be vulnerable to specific enzymatic attack, or to the hydrophobicity of the MRS to enhance or diminish fluid dissolution beyond a certain temperature or pH. Subsequent flow through the MRS, or to target the MRS to a specific site fluid diffusion or convection could effectively and irrevers or cell by coating the MRS with a particular antibody or other ibly “turn on their spectral signals. ligand. 0228 h. Far-Field Contrast Characteristics (T* Contrast 0221) f. Overall Size of MRS Agents) 0222. The size of the MRS may depend on the intended 0229. The MRS simultaneously provides frequency use of the MRS. The overall size of the magnetic resonance shifted magnetic resonance contrast, as well as a more con structure (MRS) may range from about 10 nm to about 5 cm, ventional T contrast function, by virtue of the magnetic US 2011/0014129 A1 Jan. 20, 2011 field generated by the magnetic materials of the MRS at the bottom-up fabrication methods may be compatible with a relatively far distances from the MRS in the far-field region. more limited range of materials compared to top-down meth The T contrast may be superior to comparably-sized exist ods. ing MRI contrast agents since the MRS may be constructed 0236 Particle complexes can be surface micromachined by using a top-down microfabrication method that is compat in various different ways that may, for example, include vari ible with using high magnetic moment materials. ous combinations of metal evaporation, Sputtering, electro 0230. Although the magnetic fields induced by the MRS in plating depositions, and various lithographic processes the near-field region may be essentially uniform, the external together with various wet and dry etching processes. magnetic fields in the far-field region exhibit rapid spatial 0237. In order to generate the essentially uniform mag decays in magnitude that manifest themselves as frequency netic fields used to induce the detectable and consistent fre broadened, but unshifted, background signals seen in mea quency shifts of the MRS, the geometry of the MRS is defined sured experimental spectra (for example, see FIGS. 11G-11J, as a relatively exact shape. As a result, the fabrication meth 12-15, 18D). This broadening is due to the transverse mag ods used to produce the MRS must satisfy relatively stringent netization dephasing caused by the spatially varying external conditions on the structure geometry in order to produce magnetic fields, resulting in a shortened T*. Therefore, the magnetic structures with highly consistent size and compo MRS, like any other magnetic particle, may function as a T sition. Moreover, for any embodiment in which an ensemble contrast agent. of the MRS is used, the level of inter-particle variability may 0231. An MRI image of an agarose imaging phantom be reduced to avoid any substantial broadening of the overall marked with the hollow cylindrical MRS, shown in FIG. 24, spectral signal from the ensemble. identifies the spatial locations of the structures as darkened 0238 a. Top-Down Fabrication spots that are similar in appearance to the T contrast spots 0239. The MRS may be produced using a top-down fab of existing Superparamagnetic iron oxide (SPIO) nanopar rication method. The top-down fabrication method may ticle contrast agents. This SPIO-like contrast is not Surprising include at least one spatial patterning step in the process. The given that when imaged at typical magnetic resonance spatial advantages of using top-down fabrication methods to produce resolutions, which exceed nanostructure sizes by orders of the MRS may include more directly engineered properties magnitude, a hollow shell and a solid particle present similar and increased functionality. Top-down fabrication, Such as dipolar external field profiles, and T* contrast depends only lithographic techniques, may be used to produce MRS with on magnetic moment. A comparison of the MRI image of high material purity and low variation in MRS geometry, FIG. 24 with MRI images of similarly-sized solid magnetic resulting in highly consistent frequency-shifting behavior particle contrast agents Suggests that the contrast from indi from among individual MRS. vidual MRS may be resolved within the typical resolution of 0240. The top-down fabrication method may be a micro magnetic resonance images, and that many of the dark spots machining or microfabrication method, which may fabricate shown in FIG. 24 are the contrast from individual MRS. The a structure having a size scale on the order of micrometers or MRS may therefore function as both a spatial and spectral less on a substrate. The top-down fabrication method may magnetic resonance contrast agent with magnetic dipolar also be a nanofabrication technique. The MRS may be pro magnetic fields providing spatial contrast in the far-field duced by the spatial patterning of a layer or layers of material region and essentially uniform magnetic fields within the on the Substrate using a technique Such as a lithographic reserved space providing spectral contrast within the near technique. The lithographic technique may be photolithogra field region. phy, electron beam lithography, other charged particle beam 0232 i. MRS Medium lithography, deep-UV lithography, extreme-UV lithography, 0233. The MRS generates contrast by frequency shifting and X-ray lithography. Other non-limiting examples of micro the water protons and other NMR-susceptible nuclei within a fabrication techniques include metal evaporation, ion-mill fluid medium. The fluid medium may be explicitly included ing, sputtering, micro-imprinting and nano-imprinting, elec as a part of the MRS. The MRS may additionally include a troplating, and wet and dry etching. The microfabrication medium in which one or more of the MRS is dispersed. method may be used to fabricate structures ranging in overall Non-limiting examples of a medium include a nonmagnetic size as defined above. fluid or a non-magnetic gel. The MRS may be dispersed in a 0241 The top-down method may be a resputtering tech fluid medium Such as water. nique used on photolithographically prepatterned substrates. Often regarded as an undesirable by-product of ion milling, 3. Method of Making MRS the controlled local redeposition of back-sputtered material may be exploited to yield Scalable, large-area, parallel fabri 0234. The MRS may be fabricated using any known method. Because of the strong dependence of the frequency cation of accurately defined free-standing nano structures. shifting behavior of the MRS on its geometry, the fabrication 0242. As a result of being made by the top-down fabrica methods used to produce the MRS by necessity must inher tion method, the MRS may possess low cross-structural ently produce a MRS with very low variation in MRS geom variation in size or composition. Any geometrical or compo etry or composition. Further, in order to produce a consistent sitional variation from structure to structure may induce unin frequency shift between individual MRS, the fabrication tended frequency shifts from one structure to the next. These methods should consistently produce MRS with little varia unintended frequency shifts may further cause a broadening tion from individual MRS to MRS. and degrading of the NMR spectral peaks from signals inte 0235. The MRS may be produced using top-down meth grated over ensembles of MRS. ods and bottom-up methods. Although the top-down methods 0243 (1) Top-Down Fabrication of Dual-Disk MRS produce the MRS, with typically accurate structural defini 0244. The dual-disk MRS may be fabricated using any one tion and low inter-structural variability, top-down methods of at least several top-down fabrication techniques. FIG. 3 is may be more expensive and equipment intensive to imple a schematic illustration of an exemplary embodiment of a ment. Bottom-up methods may be less expensive and equip top-down fabrication method for the production of the dual ment-intensive than top-down methods, but produce the MRS disk MRS. In this embodiment, titanium and gold layers are with a relatively higher inter-structural variability. Further, evaporated onto a wafer Substrate and a nickel/copper/nickel US 2011/0014129 A1 Jan. 20, 2011 sandwich layer may be either electroplated or evaporated on circular openings with re-entrant undercut sidewall profiles top of the gold layer at Step 1. A permanent mask layer is are patterned into a double-layer resist stack consisting of an formed on top of the outer nickel layer by spincoating, pat isotropically developing lift-off resist (LOR) beneath a nor terning, exposing, developing, and hardbaking a photoresist mal photosensitive resist layer at Step 1. To reduce undesired material at Step 2. The top nickel layer and an upper fraction lateral displacement and distortion of evaporated structures of the copper layer are ion milled through at step 3. The that result from nonperpendicular evaporation incidence copper layer is then wet-etched down to the bottom nickel angles, a thin photoresist layer of no more than 1 um thickness layer, but stopped before etching through the central copper is used and the LOR layer height reduced to just 1.25 times the Support at Steps 4. Photoresist is spincoated around the sides desired total height of the evaporated metal stack. At step 2. of the structures at step 5. The top nickel layer is used as a the base nickel, sacrificial copper, and top nickel layers are photomask so that Subsequent photoresist flood exposure and evaporated sequentially with all evaporation sources posi development leaves photoresist remaining only between the tioned directly beneath the wafer center to ensure correctly nickel layers. The spincoating at step 5 protects the top nickel overlaying metal layer alignment. The copper source may be layer and patterns the bottom nickel layer for etching. The of a larger size than the nickel source to ensure that the base nickel layer is wet etched and the internal photoresist is deposited copper layers are of slightly greater diameter than removed at step 6. The external support posts, made of SU8 the deposited nickel layers to avoid overlap of the nickel epoxy, are photopatterned at step 7, and the remaining copper layers down the side of the copper layer. As the metal depo between the nickel layers is wet etched away at step 8. sition proceeds in step 2, metal build-up around the photore 0245 FIG. 4A is a schematic illustration of another sist sidewalls shrinks the mask hole diameters. The resist embodiment of a top-down fabrication method used to pro mask is removed in step 3, yielding final circular stacks that duce an array of dual-disk MRS. In this embodiment, tita are not right cylinders but tapered conical frustums, as shown nium and gold are evaporated onto a wafer Substrate at Step 1. in FIG.38. However, the effect of this tapering is corrected for A thick layer of photoresistis spincoated and patterned at Step by depositing a thinner top nickel layer than the base layer in 2. Successive layers of nickel, copper, and nickel are electro step 2 of FIG.37. A timed copper wet-etch may be used in step plated into the photoresist mold at step 3. The photoresist 4 to form single copper central posts between the upper and mold is dissolved at step 4. A copper wet etch is initiated at lower nickel disks. Alternatively, a short selective copper step 5, and stopped in time to leave a central copper post at wet-etch may be used to expose the edges of the base nickel step 6. layer, providing contact area for external spacer posts that are 0246 FIG. 4B is a schematic illustration of yet another patterned before the remaining copper is removed, as in step embodiment of a top-down fabrication method used to pro 5. A scanning electron micrograph of dual-disk MRS result duce an array of dual-disk MRS. In this embodiment, tita ing from this fabrication process are shown in FIG. 39. nium and gold are evaporated onto a wafer Substrate and 0249 Various alternative permutations and combinations Successive layers of nickel, copper, and nickel are elacto of the steps of the exemplary top-down fabrication embodi plated or evaporated onto the Substrate at step 1. A permanent ments shown above could equally well be used to construct mask layer is formed by spincoating, patterning, exposing, dual-disk magnetic resonance structures, Solid single-disk developing, and hardbaking photoresist at step 2. The top magnetic resonance contrast agents, and any other magnetic nickel layer, copper layer, and base nickel layer are ion-milled resonance structure described above. The particular steps through followed by an angled ion-milling to remove rede selected may depend on factors including the absolute struc posited or resputtered material on the structure side walls at ture sizes and aspect ratios. Such other manufacturing tech step 3. The copper layer is wet etched partially, leaving a niques and structures made thereby are included within the central post Support at Step 4. If external Supports are desired, various top-down embodiments. SU8 epoxy support posts may be photopatterned at step 5 and (0250. The materials selected for fabrication of the MRS the remaining copper may be wet-etched away at step 6. are not limited to the materials disclosed in the exemplary 0247 FIG. 4C is a schematic illustration of still another top-down fabrication methods, but may be any magnetic and/ embodiment of a top-down fabrication method used to pro or non-magnetic material described previously. Further, the duce an array of dual-disk MRS. In this embodiment, tita MRS produced using a top-down fabrication method may nium and gold are evaporated onto a wafer Substrate and further incorporate steps to fabricate one or more coatings, Successive layers of nickel, copper, and nickel are electo including an oxidation or corrosion barrier, a mechanical plated or evaporated onto the substrate at step 1. A liftoff resist strengthening layer, a non-toxic coating, a biologically inert layer is formed by spincoating, patterning, exposing, and coating Such as titanium, or a coating to facilitate common developing photoresist at step 2. A nickel layer is evaporated bioconjugation protocols such as gold. at step 3, and the lift-off photoresist layer is removed at step 4. 0251. The various embodiments of the MRS are not lim Copper is evaporated or electroplated at Step 5, and steps 2 ited to those produced by only the top-down methods and 3 are repeated at step 6. The lift-off photoresist layer is described above or to these specific top-down methods of again removed at step 7. The copper is wet-etched at step 8. manufacture. Alternatively, another layer of patterned photoresist may be (0252 (2) Top-Down Fabrication of Hollow Cylinder MRS formed, and then the nickel layer may be ion-milled prior to (0253) The hollow-cylinder MRS may be fabricated using the wet etching of the copper in step 8. If desired, external any one of at least several top-down fabrication techniques. Support posts may be formed using similar methods to those The nanoscale lateral definition of the high-aspect-ratio walls described above. of the hollow cylinder MRS may be challenging to achieve 0248 FIG.37A-37E is a schematic illustration of an addi using the traditional top-down fabrication methods such as tional embodiment of a top-down fabrication method used to the various planar microfabrication methods described produce an array of dual-disk MRS. In this embodiment, above. The hollow cylinder MRS may be fabricated using a titanium and gold are evaporated onto a wafer Substrate and top-down technique that incorporates an unconventional US 2011/0014129 A1 Jan. 20, 2011 local resputtering of a prepatterned substrate. This local substrate sizes R that are only a few times larger than L. For resputtering fabrication method includes the novel step of example, a cosine sputter distribution gives N(z)=(N/t) ion-milling away a thin magnetic layer previously evaporated arctan(R/z)-(R/Z+z/R)'), implying a cylinder wall that onto a substrate patterned with an array of solid cylindrical deviates from its average thickness by no more than +10 posts. During the ion-milling, a fraction of the magnetic percent over the entire cylinder length for R/L values that are material emitted from the substrate redeposits on the post greater than about 7. Similarly, for a cos’ 0 sputter distribu sidewalls. By dissolving the post material, cylindrical mag tion, N.(z)=(N/t)(1+(Z/R)), implying similar wall netic nanoshells having a highly uniform cylinder wall thick thickness uniformity for R/L values that are greater than 3. ness are formed. This uniformity of cylinder wall thickness The sputtering anisotropy therefore may facilitate efficient over the full length of the cylinder results in well-defined and and parallel processing of relatively closely packed arrays of sharp NMR spectral peaks, as illustrated in FIG. 19B. structures on a substrate. However, as R/L decreases below 0254 FIG. 20A summarizes the geometry used for the the threshold values discussed above, increasingly peaked following description of the Sputter-coated wall thickness as sputter distributions and higher ion beam energies may be a function of cylinder height Z, up the side of a cylindrical necessary to maintain sufficient uniformity of the cylinder post. The Sputtered coating may naively be expected to be wall thickness. much thicker at the base of the post than at the top of the base 0258 Because excessively high beam voltages are not since points near the base are closer to the source of sputtered required to produce hollow cylindrical MRS, externally Substrate atoms than those regions higher up the post. How coated arrays of cylindrical posts may be used instead of ever, because the Sputtered atom distribution is not isotropic internally coated arrays of cylindrical holes. Although the with respect to height above the substrate, the resputtered wall internal coating of cylindrical holes may be used to produce thickness is unexpectedly uniform. According to linear colli ring-like structures, the limited Sputter target area of this sion cascade theory, sputter distributions are to first order technique implies a low effective R/L value and a resulting proportional to cos 0, where 0 is the angle between the direc substantial wall thickness variation for all but very short tion of Sputtering and the normal of the Substrate surface. cylinders. Sputter distributions have been shown to possess under-co (0259 FIG. 20B illustrates three examples of wall thick sine distributions, cosine-like distributions, and over-cosine ness variations based on solutions of Eqn. (9) for three dif distributions depending on the incident ion energies. The ferent sputter distributions. Eqn. (9) also quantifies the abso angular dependencies of the Sputter distributions may there lute wall thickness. For example, simplifying Eqn. (9) for fore be generally approximated as proportional to cos" (), R>L, a cosine sputter distribution (m=1) gives Nc/Ns 1/2. with values of m below or above unity representing under- or Assuming unit-sticking probability, the shell wall thickness is over-cosine distributions, respectively. therefore one-half of the thickness of the original layer ion 0255 Referring back to FIG. 20A, a normally incidention milled off the substrate. In this manner, the nanometer-level beam may remove N Substrate atoms per unit area or an height control common to planar thin-film layers translates equivalent amount of Nrdrdpatoms from a differential sub into similar nanometer-level width control of thin, vertically strate element P. At a distanced away from the substrate oriented Surfaces. element P, the substrate element P yields an atom fluence per 0260 Since the previously described analysis was not nec unit area of n(d) cos" 0. The proportionality coefficient n(d) essarily limited to a cylinder, other high-aspect-ratio struc =(m+1)Nsp Öpop/(2 to) may be determined by normalizing tures may be similarly fabricated. However, because some the integrated fluence through a hemispherical Surface of alternative magnetic resonance structure geometries may radius d that is centered on substrate element P. using the limit substrate visibility, locally differing limits to the R-in number of atoms emitted. Including the projection factor cos tegral and Ø-integral, and possible couplings between the Ø sin 0 to account for the angle between the atom fluence and integrals may exist. In addition, Eqn. (9) is strictly valid only the cylinder Surface normal, the number of atoms striking the for thin coatings in which t-L. For thicker coatings, the cylinderper unit area at Some representative point Q may then possibility of appreciable time-dependent modification to the be expressed as z" (m+1)-N'cos or drdo/(2C(r+z)"'), Surface normal as Substantial sidewall material accumulates, where cos 0, sin 0 and the distance PQ, are expressed interms ion erosion of the accumulated material, and reflection from of r and Z. Integrating over the half of the substrate visible accumulated material may be taken into consideration. While from point Q then gives the total number of atoms N. hitting these secondary effects may be essentially negligible for the the cylinderper unit area at height 0 coatings as described above. A selective wet-etch of the niques. Photolithographic patterning may be used to generate underlying protective copper followed by an acetone resist arrays of many of millions of solid particulate MRS to be removal then leaves the desired hollow cylinders as shown in simultaneously fabricated. A Substrate may be used to gener FIG. 21E. Each hollow cylinder at this stage is attached to the ate the solid particulate MRS. Substrate around just one half of the base, corresponding to 0267 An exemplary process of generating Solid particu the shadowed sides that did not receive any copper coating late MRS is shown in FIG. 41. A 10-nm thick titanium adhe previously, holding the hollow cylinders in place on the Sub sion layer may be evaporated onto a Supporting Substrate strate for further processing, if desired. The cylindrical shells followed by a 100-nm thick sacrificial copper layer and a may be removed from the substrate by either a gentle ultra 100-nm thick gold layer at step 1. Also at step 1, a double layer Sound treatment or a selective wet-etching of the underlying of resist may be spin-coated over the titanium-copper-gold sacrificial layer (FIG. 21F). Note that the copper layer is not trilayer with a photosensitive top layer of resist and a bottom essential in this method, but including the copper layer facili layer of isotropically developing lift-off resist. This structure tates the resist removal and provides the option of a Subse may be exposed through a mask containing an array of 2 quent water-based ultrasound release free of any metal um-diameter circular holes at step 1 as well. The patterned etchants or solvents. development and dissolution of the top resist layer results in 0262 For the case of cylindrical posts the magnetic mate the isotropic development and dissolution of those physically rial evaporation may also be performed at an oblique angle in exposed portions of the lift-off resist, creating the profile a manner similar to the copper evaporation step shown in FIG. shown in step 1. An approximately 300-nm thick layer of iron 21B, provided that the substrate is continually rotated and/or nickel may then be evaporated followed by an evapo throughout the evaporation of the magnetic material. How ration of a 200-nm thick layer of gold at step 2. The metal ever, if oblique evaporation is used to coat the post sidewalls deposited on the top of the photoresist is physically discon with magnetic material, this material may also coat the Sub nected from metal deposited on the Substrate in this step, and strate, and therefore still require Subsequention-milling, Sub subsequent removal of the resist bi-layer at step 3 may remove jecting the cylinders to similar sidewall sputter redeposition. the top metal layers while leaving the metal bilayer on the The oblique rotating evaporation of magnetic material may be Substrate untouched. A 100-nm deep argon ion-milling may conducted at shallow grazing angles relative to the Substrate, then remove the exposed gold on the substrate and about half but then the shadowing resulting from the shallow grazing of the top 200-nm gold layer at step 3. During this ion-milling angle may limit the general applicability of this technique and process Some of the back-sputtered gold ion-milled from the the spatial density of structures that may be patterned using Substrate may redeposit on the iron/nickel sidewalls, leaving this technique. Although coating the Substrate with evapo magnetic disks of nickel and/or iron completely encased in rated magnetic material may also be avoided by obliquely gold at step 4. The gold encasing the nickel and/or iron may be shadow-evaporating onto an inversely patterned array of in the form of a 100-nm thick top and bottom gold coatings, cylindrical holes rather than posts, such geometries may pre and approximately 50-nm thick gold coatings around the clude uniformly thick wall coatings. Because of the circular circumferential sidewalls of the disks. Finally a selective cylinder cross-sections, the line-of-sight penetration depths wet-etch of the underlying copper or treatment with ultra of evaporant material may vary across each hole, resulting in sound may be used to release the particles from the substrate cylindrical shells whose wall thicknesses taper down from top (not shown). The particles may also be washed to remove any to bottom. remaining etchant solution. 0263 FIG.22A is a scanning electron micrograph (SEM) 0268. These fabrications of solid particular MRS are not ofa sample array of fabricated nickelhollow cylindrical MRS limited by size, scale, dimensions, materials and various lay that have undergone a partial wet-etch release. The cylinders ers may be used as coatings and adhesion layers. have wall thicknesses of about 75 nm, cylinder inner radii of 0269 b. Bottom-Up Fabrication about 1 um, and an aspect ratio (L/2,p) of about 1.2, implying 0270. The bottom-up method may be a chemical synthesis wall height-to-thickness aspect ratios L/t of about 30. Despite technique, which may not include at least one spatial pattern having thin walls, the hollow cylindrical structures are physi ing step. The bottom-up method may use tightly-controlled cally robust, self-supporting structures that are resistant to process specifications, oran additional sorting step to select a damage during either wet-etch (see FIG.22A) or ultrasound Sub-group of MRS having acceptably similar geometric and release (see FIG.22B). In FIG. 22B, the hollow cylindrical compositional properties. structures were removed from the Substrate using ultrasound, 0271 Where only a few distinct spectral shifts induced by transferred into a vial of water, and then pipetted out onto the MRS are to be used in magnetic resonance visualization at fresh substrates. When the fresh substrates were placed into any one time, it may be possible to sacrifice Some fabrication an applied background magnetic field, the hollow cylindrical precision in order to make use of bottom-up fabrication tech MRS aligned with the applied field direction due to the anisot niques. Certain well-controlled chemical syntheses may pos ropy of the hollow cylinder's structure imparted by the high sess a high enough degree of control and monodispersity to Li?t aspect ratios of the structures, as shown in FIG.22B. provide practical fabrication methods for the MRS. 0264. These fabrications of hollow cylinder MRS are not 0272. A large batch of the MRS may be synthesized and limited by size, Scale, dimensions, materials and/or various then separated and/or filtered step to select out only those layers that may be used as coatings and adhesion layers. structures from the large batch that have geometrical shapes 0265 (3) Top-Down Fabrication of Solid Particulate that fall within a suitably narrow band of sizes and shapes. MRS-Single Disc The typically higher throughput of chemical synthesis meth 0266 The solid particulate MRS agent may be microfab ods may render this approach suitable for Some applications. ricated through the top-down method. The top-down fabrica 0273 A filtering/separation step may be accomplished by tion can include the micromachining methods of using metal taking advantage of the magnetic moment and magnetic evaporation, ion-milling and lift-off micropatterning tech materials of the MRS. For example, with a batch of structures US 2011/0014129 A1 Jan. 20, 2011 20 fabricated using a bottom-up method suspended in some magnetic resonance sensitivity by orders of magnitude, and fluid, an external magnet field gradient may be applied to reduces the required concentrations of the MRS to well below create a force on the structures that drags them through the those of existing contrast agents. The MRS may additionally fluid. In this example, the speed of the particles moving function as an individually detectable, spectrally distinct through the fluid may be governed by a balance between the micro-tag. With NMR spectral shifts determined by structural drag force of the fluid on the particles and the translational shape and composition instead of by chemical or nuclear magnetic force acting on the particles. However, the magnetic shifts, the spectral signatures associated with the MRS may and drag forces may depend on the shapes and magnetic be arbitrarily tailored over uniquely broad shift ranges span moments of the particles to differing degrees. Therefore, after ning many tens of thousands of parts per million. The MRS moving through the fluid under the influence of the applied having a size scale of micrometers may function as a localized magnetic field gradient, the differently sized/shaped/com physiological probe, enhancing both magnetic resonance posed particles may be spatially separated within the fluid, capabilities and basic biological research. The MRS may also and a Sub-group of the particles may be specifically selected be used over a wide range of applications that are analogous from the fluid based upon their location within the fluid. The to the uses of quantum dots or RFID tags. particles within this particular Sub-group may exhibit a Suit 0278 a. Dephasing Contrast Enhancement Agents ably high degree of monodispersity and may have the desired (0279 (1) Solid Particulate MRS Contrast Agents shapes. 0280. The solid particulate MRS may be used as conven 0274 The MRS may beformed using a template structure tional T contrast agent due to its magnetic materials. The Such as a porous membrane Substrate formed from a porous Solid particular MRS may be spatially imaged using the same material known in the art such as anodic alumina. The cylin dephasing contrast analysis common to MPIOS. Because the drical pores within the template structure may be filled with top-down manufacturing technique is amenable to the use of one material, the template structure may be chemically high J materials such as iron, the solid particulate MRS treated to enlarge the pore sizes, forming annular rings contrast agents possess higher magnetic moments for a given between the cylinders and the eroded template structure particle Volume compared to existing contrast agents such as within each filled pore. The annular ring may then be filled MPIOs. As a result, solid particulate MRS contrast agents with a magnetic material and the inner material may be may be used to achieve comparable contrast levels at lower chemically eroded to form hollow cylindrical structures that concentrations than existing contrast agents such as MPIOS. may be removed by again eroding the template structure by 0281 (2) MRS with Reserved Space as Dephasing Con selective chemical removal. trast Agent (0275 To fabricate the MRS, an ensemble of solid cylin 0282. In addition to acting as a conventional T contrast drical rods suspended in a solution may be chemically coated agent, an MRS with a reserved space may be differentiated with a magnetic material using a chemical method such as spectrally using the additional information provided by the electroless plating, orgalvanic deposition. For example, com NMR-shifting capabilities of the reserved space. For mercially available gold nanorods may be suspended in an example, the NMR-shift information may be used to distin electrolyte solution. However, prior to chemically coating the guish contrast signals from spurious signal Voids that con cylinders, the ends of the cylinders may be selectively chemi found magnetic resonance imaging using SPIO or MPIO cally passivated to ensure that the plating of the magnetic contrast agents. Depending on the size of the MRS, multiple material occurred only around the sides of the cylinders. The different particle spectra may be acquired simultaneously central cylinders may then be selectively etched out, leaving from a single free induction decay signal following a hard TL/2 only the plated cylindrical shell. Because typical existing excitation pulse. Alternatively, magnetic resonance imaging cylindrical rods exhibit considerable variation in diameter may spectrally resolve the tags separately, as shown for and length, an optional filtering/separation step may be per example in FIG. 11. formed as previously described to select a sub-group of hol (0283 b. MRS Identity System low cylinders having the desired shape and composition. 0284. The MRS may be used as a microtag to mark a variety of items with a unique color frequency-shift. This 4. Methods of Use frequency-shift may be measured using a MRS identity sys 0276. The MRS may be used in a variety of applications, in tem. An MRS microtag may be used to mark virtually any addition to providing magnetic resonance frequency-shifting item to which one or more MRS may be attached, or a con contrast. On a small scale, the MRS may be used to mark tainer containing one or more MRS microtags that may be various objects as a microtag or as a cell marker. The align attached. For example, MRS microtags may be used to mark ment of anisotropic MRS to an applied magnetic field may be biological cells for cell tracking studies, packages for track used to determine the direction or other characteristics of a ing during shipping, and inventory for industrial inventory flow. On a large scale, the MRS may be installed around the control. perimeter of a variety of fluid-carrying vessels ranging Such 0285 FIG. 5 is a system diagram of an embodiment of a as microfluidics channels or blood vessels and used to fre magnetic resonance identity system 200. The magnetic reso quency-shift the fluid passing through the reserved space of nance identity system 200 includes at least one MRS 202, a the MRS, providing spin-tagged flow for magnetic resonance source of electromagnetic radiation 204 to illuminate the flow visualization. magnetic resonance microstructure 202 with an excitatory 0277 Shifts in the Larmor frequency of water protons or electromagnetic pulse, and a detection system 206 to detect other NMR-susceptible nuclei within a near-field region of electromagnetic radiation emitted from within the magnetic the MRS during exposure to a resonant electromagnetic pulse resonance microstructure 202 after the MRS 202 has been may be used to conduct multiplexed color magnetic reso illuminated with the an excitatory electromagnetic pulse. The nance visualization. Engineered to exploit diffusion and/or MRS 202 may be a solid particulate MRS that functions fluid flow in some embodiments, the MRS increases existing solely as a T contrast agent, or an MRS with a reserved US 2011/0014129 A1 Jan. 20, 2011 space that may act as either a T contrast agent, a NMR embodiments of the current invention to provide redundancy, shifting contrast agent, or both. The magnetic resonance iden to be used for alternative blood flow speed measuring (for tity system 200 may also include a magnetic field generation example via time-of-flight techniques), or to increase the system 208 to provide a magnetic field in a region suitable for contrast signal magnitude. A similar measurement of blood the placement of a sample of interest that may include the flow within a blood vessel may be non-invasively measured MRS microtag. without a stent device by placing the magnetic elements of the 0286 c. MRS Stent MRS arrayed around the outside of a vein/artery or other 0287. A stent that includes an MRS with a reserved space blood vessel to monitor blood flow within that blood vessel. may be used to monitor blood flow through the stent as well as 0294 d. Spin-Tagging Fluid Flow/Perfusion Imaging remotely monitoring the condition of the stent. Discrete vol 0295 MRS may be situated such that a fluid flows through umes of NMR-shifted waterprotons created at different times the reserved space, and a volume of fluid within the reserved within the reserved space may be visualized using NMR space may be frequency-shifted by the uniform magnetic imaging and used to estimate the blood flow speed down field of the MRS. For a limited period of time after leaving the stream of the stent. In addition, the magnitude of the NMR reserved space, the volume of fluid may retain the shifted shift may measured and used to determine changes in the NMR frequency, effectively spin-tagging the fluid as it flows. condition if the stent collapse or their is distortion of the stent. Using magnetic resonance visualization methods described 0288 The MRS may be installed around the inner or outer above, the spin-tagged fluid may be visualized and analyzed perimeter of a stent in order to measure the characteristics of to determine a variety of flow characteristics such as flow blood flow through the stent. Alternatively, the stent may be speed. entirely composed of the MRS structure. The stent is situated 0296 To perform the spin-tagging of fluid flow, one or such that the blood flow passes through the reserved space of more MRS may be situated around the perimeter of a fluid the MRS. vessel and/or along the length of a fluid vessel and the fluid 0289 FIG.25 is a schematic illustration of an embodiment flowing through the reserved space of the MRS may be fre of a hollow cylindrical MRS that functions in conjunction quency-shifted and visualized using magnetic resonance with a stent. In this embodiment, a thin ring-like magnetizable techniques to provide non-invasive visualization of fluid flow. solid structure surrounds or is attached to the inside walls of This concept of spin-tagging fluid as it passes through the a stent device. The MRS of this embodiment may also be one uniform magnetic field within the reserved space of the MRS or more dual-disk MRS, as shown in FIG. 26. If more than one structures may be used at a variety of size scales ranging from dual-disk MRS are used, each pair of disks may be situated at vessels that are about 1-um, 2-um. 5-lum, 10-um, 20-lum, the same longitudinal position along the stent, but rotated 30-um, 40-um, 50-um, 60-um, 70-um, 80-lum, 90-um, 100 around the stent's axis of symmetry by different rotations um, 200-lum, 300-um, 400-um, 500-um, 600-um, 700-um, angles. There may also be a plurality of dual-disk MRS 800-um, 900-um, 1 mm, 2 mm, 3 mm, 4 mm, 5 mm diameter arrayed around at differentangles to approximate a ring struc to vessels that are about 5 cm or more in diameter. Fluid flows ture that may be visualized using MRS spin-tagging methods may 0290 The particular magnetic resonance structure may be include perfusion and other blood flow, industrial fluid flow, selected based on the type of stent in which the structure is to and flow in microfluidic systems. For example, applications be used. For example, a dual-disk geometry may be more may include measuring, imaging, or detecting flow within a favorable geometry if the stent device is intended to be microfluidic channel or network as may exist in various inserted in a collapsed position and then expanded via a microchip-based chemical and biological assays (i.e., lab-on catheter balloon after the stent is situated in its intended a-chip systems). As another example, the flow in industrial location. pipes or pipelines may be visualized using spin-tagging of the 0291. The spectral shifting within the reserved space of fluid within the pipes using one or more MRS that may be the MRS allows the water protons or other NMR-susceptible arrayed externally about the exterior circumference of the nuclei in the blood flowing through the MRS to be spin pipes, or contained within the pipes or attached to the inner labeled so that blood flow (both speed and, through fre walls of the pipes. Spin-tagging using MRS may further pro quency-shift-dependent stent diameter indications, mass vide flow monitoring capabilities even if the pipes are non flow) can be measured. Such spin-labeling, alternatively also transparent. Flow monitoring capabilities may include known as spin-tagging, can be performed by, for example, observing where fluid Subsequently flows, measuring the irradiating the MRS with resonant RF electromagnetic pulse flow speed, and how the flow speed varies across one or more to specifically spin-tag NMR-susceptible nuclei within the cross-sections of the pipe or along the length of the pipe. reserved space. Fluid not resident within the reserved space 0297. The MRS may be used to spin-tag discrete volumes during a resonant RF electromagnetic pulse would be essen of fluid containing NMR-susceptible nuclei such as water tially unaffected by the pulse. protons by exposing the MRS to discretely spaced electro 0292 Should the artery or other blood vessel containing a magnetic pulses at the resonance frequency of the MRS. Fluid stent narrow, the stent diameter may shrink as a result, caus contained within the reserved Volume during each resonant ing the NMR frequency shift to be altered, as discussed above electromagnetic pulse is phase-shifted, and any remaining and shown in Eqn. (2) and/or Eqn. (7), due to the change in fluid outside of the reserved volume is unaffected by the spacing between magnetizable elements. A similar effect may resonant electromagnetic pulse. Fluid flowing downstream of occur if the stent itself was in Some way damaged or started to the MRS that has been phase-shifted in this may be visualized collapse. Thus, the inclusion of MRS within a stent device using magnetic resonance visualization. FIGS. 28 and 29 are enables the non-invasive NMR measurement of artery col MRI images showing the bands of spin-tagged fluid down lapse or warning of possible imminent stent collapse. stream of an MRS. In FIG. 28, both pipes in the figure were 0293. There may also be multiple NMR spaced at pre exposed to a discrete series of electromagnetic pulses at the determined intervals longitudinally along the stent in some resonant frequency of the left MRS prior to MRI scanning. In US 2011/0014129 A1 Jan. 20, 2011 22 FIG. 29, both pipes were exposed to a discrete series of cal instrument may be individually tracked and guided using electromagnetic pulses at the resonant frequency of the right the multispectral magnetic resonance visualization methods MRS prior to MRI scanning. Each dark band in the pipes described above. Further, cells or tissues to be targeted by the mark the parabolic profile of a spin-tagged Volume that has Surgical instruments may be marked with yet another group of traveled in a laminar flow within the pipe for a short distance MRS having another NMR-shift signal frequency to provide downstream from the reserved volume. In addition, because a target for the Surgical instruments using multispectral mag the right hand pipe in FIG. 28 was not exposed to an electro netic resonance visualization. magnetic pulse at its resonant frequency, which is different 0304. An activatable MRS, described above, may be from the resonant frequency of the left MRS, none of the flow attached to a moving Substrate such as a Surgical instrument in the right pipe was spin-tagged, resulting in a uniformly and used as a smart sensor in which the NMR frequency shift light image in the MRI image. The flow in the left pipe in FIG. changes as a function of some physiological condition Such as 29 was not spin-tagged for similar reasons. temperature, oxygen content, or pH. For example, as the 0298. Additionally, the MRS may be used to conduct per moving substrate is moved within the field of view of the fusion studies with multiple spin-labeled streams that are NMR visualization device. Such as during a Surgical proce immune to magnetic mixing. Such a process may also be dure, the NMR frequency shift of the MRS may be monitored applied to perfusion imaging where the resonant electromag to assess one or more physiological conditions in order to netic labeling pulses are spaced close enough in time so as to monitor the conditions of a Surgical procedure or to guide the appear continuous in an image, as shown in FIGS. 30 and 31 placement of the Surgical instrument. for the left and right tubes, respectively, where in this case the (0305 f. MRS Microtags and Specific Detection/Labeling/ spin-labeled flow occupying half of each tube is darkened. Tracking of Biological Cells For example, this technique may be used to show where fresh 0306 One or more MRS microtags may be affixed to an blood enters the brain and perfuses. object, allowing that object to be magnetically probed and/or 0299 The spin-tagging technique of flow visualization recognized using the magnetic resonance visualization tech may be used to measure the features of both laminar Poi niques described above. Unique combinations of MRS about seuille flow, as well as turbulent flow. Finer flow features such 1-um to about 1-mm in overall size may be used to label an as turbulence, Vortex structure, or boundary layer structure object Such as a cell, organism, or non-biological object for may be measured using spin-tagging, so long as the magnetic identification using magnetic resonance Scanning. The iden resonance visualization device used to visualize the spin tification information may be encoded by the combinations of tagged flow possessed sufficient resolution. MRS in a manner analogous to RFID-tagging. In addition, by 0300 e. Magnetic Resonance Spatial Calibration Mark marking each object with one or more different MRS particles ers/Locators (when Affixed to Substrate) having different frequency shifts, different objects may be 0301 An array of MRS with known separation distances distinguished from each other in much the same way as regu and angles may be used as a calibration aid for a magnetic lar RFID chips do by marking the object with one or more resonance device. A set of the MRS might be arrayed in some MRS having a specific known NMR frequency shift, except regular geometrically prescribed arrangement with known that the identifying signal is based on a nuclear magnetic spacings and/orangles between the individual MRS in the set, resonance measurement. For example, objects of type A may firmly attached to a rigid substrate to provide a spatial cali be marked with a MRS having a NMR frequency shift A, and bration of measured distances and angles in a magnetic reso another object B may be marked with a different MRS having nance device. If MRS with reserved spaces are used, MRS a NMR frequency shift B. with two or more frequency-shifting characteristics may be (0307. The MRS particles and the objects that they label placed in close proximity within the set, even within the same may reside within a fluid, gas, or gel Suitable for magnetic Voxel of the magnetic resonance device, so long as the MRS resonance probing, or the MRS particles may be packaged in are separated by at least about twice the maximum dimension a separate container along with Some amount of fluid, gas, or of the MRS. Using multi-spectral scanning methods, in which gel, and the entire container and MRS particles contained the calibration aid is imaged after each exposure to electro within may be affixed to the object as a marker. The MRS magnetic pulses at each of the resonant frequencies of each tagged object need not itselfbe within the fluid or gel in order subset of the MRS in the calibration aid. A much higher to be marked. calibration resolution may be achieved than is possible using 0308 One or more MRS may be bound to or incorporated MRS with uniform contrast properties or existing magnetic within certain biological cells to mark the cells for subsequent particle contrast agents. magnetic resonance visualization studies. In this example, the 0302) In addition, the MRS may be attached to a moving MRS might include a specific biochemical coating ensuring Substrate within the field of view of an MRS device. For that the MRS specifically binds to a specific cell type. This example, an MRS may be attached to the tip of a surgical would enable tracking of cells and in particular, the ability to instrument such as a catheter, and magnetic resonance visu differentiate between different cell types by exploiting the alization may be used to track the location of the Surgical different frequency shifts of the attached MRS. instrument non-invasively and/or guide the Surgical instru 0309. A biological cell labeled with several solid-disk ment during a Surgical procedure. MRS attached to the outer cell membrane is shown in FIG.52. 0303 If an MRS with a reserved space is attached to a A biological cell labeled with several solid-disk MRS that surgical instrument or other moving substrate, the NMR have been incorporated into the cytoplasm of the cell is shown shifting signal of the MRS may be used to identify the par in FIG.S3. ticular moving substrate as it moves within the field of view of 0310. The objects marked using MRS microtags may be in the magnetic resonance device. Each of two or more Surgical placed in stationary containers, or the objects may be situated instruments may be marked with MRS with different NMR within a vessel containing a moving flow of a fluid. For shifting signal frequencies and the movements of each Surgi example, MRS microtags may be used to label objects flow US 2011/0014129 A1 Jan. 20, 2011 ing in a microfluidic steam, so that remote sensing and iden 0316 Alternatively, a uniform geometrical array of MRS tification of the labeled objects may be made as they are particles may be arranged with the geometry of each MRS transported within the microfluidic steam. Because this particle varied such that each frequency shift differs by a method makes use of magnetic resonance visualization tech predetermined amount from the frequency shift of the neigh niques, an optical line of sight is not required to identify the boring MRS particles. Within the array, neighboring MRS objects, unlike existing microfluidics identification methods. may be spaced at least about 2-3 times the maximum outer As a result, this method may also be useful for monitoring dimension of the MRS away from all neighboring MRS to microfluid flows in otherwise inaccessible locations within a minimize the interaction of neighboring MRS external mag microfluidics device. netic fields. 0311. In another example, living biological cells may be 0317. A magnetic resonance image of this array would labeled using one or more MRS microtags functionalized show higher or lower signal amplitudes at a specific location with specific antigens or other binding agents in order to label in the array due to the frequency-shifting effects of the exter particular flow types. In this example, flow cytometry may be nal magnetic field. Using this MRS particle array, the mea conducted by inducing the cells to flow past a magnetic reso surement of the magnetic field is effectively transformed nance sensor. Alternatively, living cells Such as blood cells from a field measurement method into a method of visually may be labeled as they circulate using one or more MRS locating the spatial position of the higher or lower signal, and microtags, and in vivo flow cytometry may be performed by determining the field strength from the known frequency sensing the labeled cells using a magnetic resonance scanner shifting characteristics of the MRS particle at that location. focused in a specific region of a blood vessel of a living 0318 h. Distance/Pressure/Vibration/Torque Sensors (all subject. In yet another example, individual labeled cells may will Affect the Particles Measurable Frequency Shifts betracked as they move within the circulatory vessels or other through Change in Particle Geometry) tissues or organs of a living Subject. 0319. The MRS may be designed so that the frequency 0312. The overall size of the MRS used to label living cells shifting behavior may depend on a physical factor Such as ranges from about 1 um to about 10um, or may be about 1 um, pressure, vibration, orientation changes, or torque experi 2 um, 3 Jum, 4 Jum, 5um, 6 Jum, 7 um, 8 um, 9 Lim, or 10 Lum. enced by the MRS. Magnetic resonance visualization of an The minimum overall size is limited to the smallest particle MRS with this design may be used to non-invasively assess that is still visible using nuclear magnetic visualization, and physical forces withina living Subjector within another struc the maximum overall size is limited to the largest particle that ture or fluid flow. Because the frequency-shifting of the MRS may be placed in the cytoplasm of a living cell without depends on, among other factors, the spacing between the adversely affecting the viability of the labeled cell. In addi magnetic portions and the orientation of the MRS relative to tion, in order to be detectable by a magnetic resonance device, the background magnetic field, an MRS may be used to the MRS microtag must have a magnetic moment of at least measure a variety of physical phenomena by transducing about 10 Am. The MRS microtags may be affixed to the these phenomena into a distance change between the mag outer cell membrane or cell wall, or the MRS microtags may netic portions. be inserted into the cytoplasm of the labeled cell. The MRS 0320 For example, an MRS may be designed to have a microtags may be introduced into the cell by endocytosis diminished ability to self-align to an applied magnetic field means such as phagocytosis, macropinocytosis, caveolae, direction. Because the frequency-shifting behavior of the clathrin-mediated endocytosis or receptor-mediated endocy MRS also depends on its alignment with an applied magnetic tosis endocytosis. MRS microtags may also be introduced by field, changes in the frequency-shifting signal from an MRS genetic engineering methods such as electroporation or pro with this design may be used to assess the degree of alignment toplasts. with the external magnetic field. By altering the self-aligning 0313 Apart from identifying or tracking labeled objects behavior relative to the tendency to align with other applied moving in stream, this method may also be used to infer forces such as fluid dynamic torques, the strength of the additional information about the fluid stream, such as flow frequency-shifting signal may be used to measure the mag speed by noting how the MRS microtags move within the nitude of the other applied forces. Stream. 0321) Such orientation sensing may also be used to map 0314 g. Magnetic Field Sensors fluid flow direction or for measuring fluid flow strength. For 0315. An array of MRS particles in which each MRS example, if the fluid dynamic forces were stronger than the particle has a slightly different frequency-shifting behavior magnetic self-alignment forces, then the orientation of the may be placed within a magnetic field in order to measure its MRS, as measured by the strength or existence of the char strength. Because the resonance frequency of the MRS deter acteristic spectral signature of the MRS, may depend on the mines an offset in the Larmor precession frequency of the fluid flow direction relative to the direction of the applied nuclear magnetic moments that pass through the reserved magnetic field. Vasculature network geometries that may be space of the MRS, the exact absolute resonant Larmor pre too small to be visualized using existing magnetic resonance cession frequency induced by the MRS may be used to pro techniques may be mapped using the magnetic resonance vide a visual measure of the total magnetic field created by the visualization of an MRS with this design. In addition, fluid Superposition of the magnetic field within the reserved space flow strength may be measured by observing whether or not and the external magnetic field. Because the magnitude of the the applied magnetic field is Suitably strong to realign an magnetic field within the reserved space may be determined MRS situated within a fluid flow. from the geometry and materials included in the MRS, the 0322. Different portions of the magnetic material within magnitude of the external applied magnetic field may be an MRS may be designed to change orientation with respect determined by subtracting the known magnetic field from the to each other in reaction to an applied force or torque. The reserved space from the total magnetic field deduced from the change in the relative orientation of the different portions of frequency shift induced by the total magnetic field. the magnetic materials alters the frequency-shifting of the US 2011/0014129 A1 Jan. 20, 2011 24 MRS with this design. The changes in frequency-shifting that specifically bind only to cancerous cells, normal cells behavior of the MRS, as measured using magnetic resonance would effectively be unharmed using this method. The MRS visualization methods, may be used to provide an indirect in this example may be attached to the outer surface of the cell measure of one or more torque forces acting on the MRS, or membrane or cell wall, the inner surface of the cell mem simply a different angular orientation of the MRS. brane, or may be inserted into the cytoplasm of the cell to be 0323 For example, if a double-disk MRS with one disk destroyed. fixed to a rigid surface is placed into flow of sufficient veloc 0332 The strength of the rotating magnetic field used to ity, the shear forces of the moving fluid acting on the unat rotate the MRS may range from about 1 Gauss to 1 Tesla, tached disk may exert a force that displaces the free disk depending on the size and magnetic moment of the MRS relative to the immobilized disk, causing the NMR frequency used. The overall size of the MRS used to destroy selected shift signal to cease. The system may be calibrated Such the cells using this method may ranges from about 1 Lum to about MRS stops producing a NMR frequency-shift signal at a 10 um, or may be about 1 lum, 2 um, 3 um, 4 um, 5um, 6 um, known flow speed or shear force, or any array of MRS in 7 yum, 8 um, 9 Lim, or 10 Lum Localized RF magnetic heating which each MRS having a different NMR frequency-shift elements/targeted thermal ablation stops producing NMR frequency-shift signals at a different 0333 Depending on the exact material composition of the predetermined flow speed or shear force. magnetic portions comprising these microstructures, applica 0324. In another example, an MRS may be designed to tion of an alternating magnetic field that repeatedly magne measure fluid pressures in the blood stream. In this example, tizes and demagnetizes the objects could be used to generate the alignment of the MRS with respect to the magnetic reso local heating for targeted thermal ablation/destruction of bio nance magnetic field may be governed by an equilibrium logical cells such as cancerous cells. between the magnetic self-alignment torques of the MRS 0334] Any non-spherical shape of MRS may be used to from the magnetic resonance magnetic field and the rotational destroy selected cells using this method, including disks, and/or shear forces exerted by the flowing fluid. rods, and cylinders. Any shape of MRS that includes a sharp 0325 In yet another example, two or more MRS having edge may be particularly effective in destroying selected different frequency-shifting characteristics may be attached cells. to different locations along an object such as a protein mol 0335 k. Localized Magnetic Field Gradients ecule. The distance between the two different MRS may be 0336. The MRS particles may be used in alternative mag estimated using the NMR multi-spectral imaging data. If the netic imaging techniques that take advantage of the relatively two MRS move to within 2-3 times the MRS size of each high localized magnetic field gradients external to each MRS. other, the NMR-shifting signals cancel each other out due to The external magnetic fields of the MRS particles produced the mutual interference of the external magnetic fields of the using high JS materials such as iron may induce exceptionally two MRS. Thus, very small distances may be detected using high magnetic field gradients that may be useful for alternate the disabling of the MRS signal, in a manner analogous to magnetic imaging techniques, or for generating highly local fluorescence resonance energy transfer (FRET) measurement ized high magnetic forces. techniques. 0326 In another example, a fluctuation in the frequency Examples shift magnitude may be used to sense vibrations using any of the MRS configurations described above. Example 1 0327 i. Magnetic Separation 0328 Being magnetic, these microstructures could be Assessment of Saturation of Dual-Disk Magnetic used in the same manner as regular magnetic beads in tradi Resonance Structures tional magnetic separation protocols. 0337 To assess the effect of the magnitude of applied 0329 j. As Rotators of Objects Attached to them/Magneti magnetic field on the magnitude of the induced magnetic field cally Driven Rotary Pump-like Motion/Fluid Pump/Mixer in the reserved Volume of a magnetic resonance structure, the 0330. The MRS particles may be rotated indirectly using a following experiment was conducted. A 13 mmx13 mm grid rotating externally applied magnetic field and used to act as of immobilized dual-disk magnetic resonance structures was micropumps or micromixers in a variety of systems. A MRS tested using an alternating gradient magnetometer to assess particle may be designed with a high degree of magnetic the magnitude of the magnetic field induced within the shape anisotropy, resulting in a relatively high self-aligning reserved Volumes as a function of the magnitude of the magnetic torque between the MRS particle and an external applied magnetic field, as well as the hysteresis of the induced applied magnetic field. By applying a rotating magnetic field magnetic field during a full alternating gradient cycle. Each to a MRS particle with a high magnetic anisotropy, a strongly dual-disk magnetic resonance structure in the 13 mmx13 mm rotating MRS particle results. This rotating behavior could be grid was formed on a 15mmx15 mm diced Pyrex substrate exploited to make fluid micropumps and micromixers. using microfabrication techniques. Each disk in a dual-disk 0331. Such rotation may also be useful for destroying magnetic resonance structure was a pure nickel disks having selected biological cells such as cancerous cells by placing a disk radius of about 2.5um, a thickness of about 50 nm, and these microstructures within Such cells and then rotating the a disk separation distance of about 2 um. The nickel material external field to effectively churn up the cell's contents. The from which the disks were constructed had a saturation mag MRS may be introduced into the cell by endocytosis means netic polarization (J) between about 0.5 and about 0.6 Tesla. Such as phagocytosis, macropinocytosis, caveolae, clathrin Inter-particle spacings (center-to-center) were typically 3 to 4 mediated endocytosis or receptor-mediated endocytosis times the particle diameter to minimize the influence from the endocytosis. The MRS may also be introduced by genetic induced far-field magnetic fields of neighboring particles on engineering methods such as electroporation or protoplasts. the induced magnetic field of each individual magnetic reso If the MRS in this example were coated with binding agents nance structure in the grid. US 2011/0014129 A1 Jan. 20, 2011 0338 A magnetic field was applied to the grid of magnetic particle localization. The particle spectra of each particle resonance structures that alternated between a value of subgroup, as shown in FIGS. 11G-11J were shifted well away +2000x10/4t A/m, and the magnetic polarization of the from the unshifted water proton line. Further, as shown in dual-disk magnetic resonance structures was assessed. FIG. 11J, the particle spectra are each sufficiently separated, 0339. The results of the alternating gradient magnetom allowing for the unambiguous color-coding of the individual eter measurements are summarized in FIG. 7. The dual-disk particle types with minimal background interference. magnetic resonance structures achieved a saturated magnetic 0345 The results of this experiment demonstrated the fea polarizationatan applied magnetic field of about it 1000x10/ sibility of using the dual-disk magnetic resonance structures 4JLA/m, well below the magnitude of applied magnetic field to achieve multispectral magnetic resonance imaging using a generated by typical magnetic resonance scanning devices. chemical shift imaging (CSI) process. 0340. The results of this experiment confirmed that the dual-disk magnetic resonance structures achieved a saturated Example 3 magnetic polarization at applied magnetic field magnitudes that are well within the capabilities of existing magnetic Frequency-Shifting of Deuterium Proton Signals by resonance scanning devices. As such, the field shift are inde Dual-Disk Magnetic Resonance Structures pendent of the MRI fields. 0346. To determine the effectiveness of the frequency shifting of protons other than water protons, the following Example 2 experiment was conducted. A grid of dual-disk magnetic resonance structures similar to those described in Example 1 Multi-Spectral Imaging Using Dual-Disk Magnetic were submerged in deuterium oxide (DO) and imaged in a Resonance Structures manner similar that described in Example 2. In this experi 0341 To assess the effectiveness of multispectral imaging ment, the disks in the dual-disk magnetic resonant structures using magnetic resonance structures in a magnetic resonance had a diameter of about 25um, a disk thickness of about 0.5 scanning device, the following experiment was conducted. A um, and a separation distance of about 10 um between the grid of immobilized nickel dual-disk magnetic resonance disks. A grid of dual-disk magnetic resonance structures was structures were fabricated in a manner similar to those constructed similar to those described in Example 1. described in Example 1. The disks in each dual-disk magnetic 0347 An individual pyrex chip was placed in a custom resonance structure in this experiment had a diameter of about made holder and filled with a layer of deuterium oxide (DO) 1.25 mm and a separation of about 500 mm between the disks. to a thickness of about 150 um, in order to submerge the Inter-particle spacings (center-to-center) were typically 3 to 4 particles and provide an additional layer of deuterium oxide times the particle diameter to minimize the influence from the (DO) well above the extent of any appreciable external mag induced far-field magnetic fields of neighboring particles on netic fields induced by the magnetic resonance structures. the chemical shifting of each individual magnetic resonance The deuterium oxide (DO)-submerged pyrex chip sample structure in the grid. Subgroups of the dual-disk magnetic was then placed next to or inside of the surface or solenoidal resonance structures had disk thicknesses of 4, 6, and 8 um coils of the magnetic resonance scanning device for transmis respectively in order to vary the frequency shift induced by sion/reception of the NMR signal. each respective subgroup. Each Subgroup was arranged on the 0348 Free induction decay (fid) signals following a spin grid to form the letters R, G, or B, as illustrated in FIG. 11A. echo pattern were acquired in a manner similar to that Accidental impurities in the nickel discs of these structures described in Experiment 2. The bandwidth of the measure led to a reduction in the Saturation magnetic polarization (J) ments was limited to about -75 kHz due to the limitations of of the disks to about 0.4T. The disks in each dual-disk mag the measurement coil. netic resonance structure in this experiment were Submerged 0349 The spectrum obtained from the measurements in water. described above is shown in FIG. 12. The magnetic resonance 0342. Free induction decay (fid) signals following a spin structures induced a well-defined frequency shift of the deu echo were acquired Sweeping through a range of frequencies terium protons of about -50 KHZ. This frequency shift spec covering the expected offsets produced by the particles. trum is in good agreement with estimated theoretical values. Shaped pulses with a Gaussian profile were used to limit 0350. The results of this experiment demonstrated the bandwidth spread into the bulk water peak (as compared to a ability of the magnetic resonance structures to frequency hard pulse). The bandwidths were sufficient to cover the shift deuterium oxide protons as well as water protons. frequency profiles produced by the particles. Acquisitions for the spectra were 8192 points in length, covering a bandwidth Example 4 of-100 kHz. For the associated RGB image, three 2D chemi cal shift images were acquired, covering the frequency ranges Effect of Pulse Delay on Frequency Shifting by of the particle spectra. Images are integrations of the spectra Dual-Disk Magnetic Resonance Structures over the different frequency ranges. In-plane resolution was 0351. To assess the effect of the timing of preparatory 500x750 um. off-resonance pulses on the frequency shifting of water pro 0343 AN image of the grid obtained using gradient-echo tons and other NMR-susceptible nuclei by magnetic reso (GRE) MRI is shown in FIG. 11B. Magnetic dephasing due to nance structures during magnetic resonance scanning mea the effects of the far-field magnetic fields induced by the Surements, the following experiment was conducted. particles enables the spatial imaging shown in FIG. 11B. 0352. A grid of dual-disk magnetic resonance structures 0344 FIG. 11C-11E show the chemical shift imaging similar to those described in Example 3 were submerged in (CSI) of the grid magnetized by an applied magnetic field Bo. water instead of deuterium oxide (DO) in a manner similar to The additional spectral information provided by CSI imaging that described in Example 3. In this experiment, the disks in differentiates between individual particle types and improves the dual-disk magnetic resonant structures had a diameter of US 2011/0014129 A1 Jan. 20, 2011 26 about 5um, a disk thickness of about 65 nm, and a separation technique. In one grid, the disk diameter was about 5um, the distance of about 2 um between the disks. thickness of each disk was about 50 nm, and the disk separa 0353. The submerged grid of particles was subjected to tion distance was about 2 um. In the other grid, the disk magnetic resonance measurements using an indirect detec diameter was about 3 um, the thickness of each disk was about tion technique. The magnetic resonance device delivered a 50 nm, and the disk separation distance was about 1 lum. series of off-resonance pulses (Gaussian shape, 100 us in 0359 The Z-spectra obtained from the two grids of mag length) for a period of a few T’s, followed by an on-reso netic resonance structures is shown in FIG. 15. The grid nance 90-degree pulse, then the collection of fid data. Each containing the Smaller-radius dual disk magnetic resonance point in a Z-spectra was calculated by integrating the fid data structures had a higher frequency shift compared to the for each different off-resonance frequency of the preparatory larger-radius grid. The frequency shifts of the two magnetic pulse train. The gap between each pulse in a preparatory pulse resonance particles measured in this experiment were suffi train was varied between 1 ms and 5 ms. ciently separated in frequency shift (about -370 kHz vs. 0354. The Z-spectra obtained for pulse train gaps of 1 ms, about -200 kHz), and possessed sufficiently narrow line 2 ms, and 5 ms are shown in FIG. 13. Closer spacing of the width to ensure the detection of individual signals in a mul preparatory pulses resulted in a higher magnitude of signal at tiplexed magnetic resonance measurement technique. the shifted frequency, with negligible effect on the amount of 0360. The results of this experiment demonstrated that frequency shift. variation in the radii of the disks in a dual-disk magnetic 0355 The results of this experiment determined that that resonance structure resulted in detectably distinct frequency the frequency shift induced by the magnetic resonance struc shifting by the magnetic resonance structures in a multiplexed tures is insensitive to the spacing of the off-resonance prepa magnetic resonance measurement environment. ratory pulses. However, the amount of frequency-shifted water protons and other NMR-susceptible nuclei, as indi Example 7 cated by the magnitude of the fid signal at the shifted fre quency, increases when the preparatory pulses are spaced Effect of Disk Thickness on Frequency Shifting by closer together. Dual-Disk Magnetic Resonance Structures 0361. To assess the sensitivity of the frequency shift Example 5 induced by a dual-disk magnetic resonance structure to varia Example 5 tion in the thickness of the disks, the following experiment was conducted. Grids of dual-disk magnetic resonance struc Effect of Applied Magnetic Field Strength on Fre tures Submerged in water similar to the grid described in quency Shifting by Dual-Disk Magnetic Resonance Example 4 were measured using a similar indirect detection Structures technique. Each grid contained an array of dual disk magnetic 0356. To determine the effect of the applied magnetic field resonance structures with the same specified disk thickness; strength on the frequency shifting of water protons and other the specified disk thickness of the different grids varied NMR-susceptible nuclei by magnetic resonance structures between about 50 nm to about 75 nm. during magnetic resonance scanning measurements, the fol 0362 FIG.16 is a summary of the Z-spectrum values mea lowing experiment was conducted. The grid of dual-disk Sured for each of the grids having disks with varying thick magnetic resonance structures Submerged in water described nesses. Each row in FIG. 16 shows the experimental H0 in Example 4 was measured using a similar indirect detection Z-spectrum for a different particle disc thickness. In this fig technique. In this experiment, an identical preparatory pulse ure, the raw Z-spectra of the shifted peaks atop the unshifted sequence was used for each set of measurement. However, the broadened waterbackground is shown. This background may sets of measurements obtained in this experiment were con be eliminated by calculating the differences between corre ducted using magnetic field strengths Bo of 4.7T, 7.0 T, and sponding positive- and negative-frequency Saturation signals 11.7 T. Differing magnetic field profiles from the different to eliminate the effects of the water background signal. coils used may have introduced limited variability in the 0363 As shown in FIG. 16, the dual-disk magnetic reso results. nance structures induced a frequency shift of about -360 kHz 0357 The Z-spectra for the fid signals induced by the at a disk thickness of 50 nm, which increased gradually to a magnetic resonance structures are shown in FIG. 14. Varia frequency shift of about -500 kHz at a disk thickness of 75 tion in the applied magnetic field strength did not signifi nm. In addition, as the thickness of the disks decreased, the cantly alter the frequency shift induced by the magnetic reso line width of the frequency shift became increasingly broad. nance structures. At the shift frequency, the fid signal was 0364 The results of this experiment demonstrated that the higher in magnitude at the higher applied magnetic field magnitude of the frequency shift induced by a dual-disk mag strengths. netic resonance structure may be predictably and controllably manipulated by varying the thickness of the disks. Example 6 Example 8 Effect of Disk Radius on Frequency Shifting by Dual-Disk Magnetic Resonance Structures Effect of Asymmetries of Dual-Disk Magnetic Reso nance Structures on Induced Frequency Shifting 0358 To assess the sensitivity of the frequency shift induced by a dual-disk magnetic resonance structure to varia 0365. To assess the effects of asymmetries within a dual tion in the radii of the disks, the following experiment was disk magnetic resonance structure on the frequency shift conducted. Two grids of dual-disk magnetic resonance struc induced by the structure, the following simulations were con tures Submerged in water similar to the grid described in ducted. Numerical calculations were performed to estimate Example 4 were measured using a similar indirect detection the effect of various asymmetrical variations in the geometry US 2011/0014129 A1 Jan. 20, 2011 27 of a dual-disk magnetic resonance structure on the structure's similar to the grid described in Example 4 were measured frequency shift characteristics. All calculations were per using a similar indirect detection technique. In this experi formed for a dual disk structure having a saturated magnetic ment, the spaces between the disks of a portion of the struc density of 0.6T (corresponding to nickel), a disk diameter of tures were filled in, as shown in SEM image in the left-hand 2um, a disk thickness of 40 nm, and a disk separation distance inset of FIG. 17. The right-hand inset figure of FIG. 17 is a of 0.85um. picture of animage obtained by an magnetic resonance device 0366 One set of calculations varied the radius of one disk using an indirect detection technique. The group of structures of the pair from 100% to 65% of the value of the other disk. with filled-in reserved volumes (“OFF' group) did not gen The results of these calculations are summarized in FIG. 32. erate a signal, and the group of structures which had open As the disks become increasingly different in size, there is a reserved volumes (“ON” group) to allow the diffusion of fluid significant signal loss, peak broadening, and alteration of the in and out of the reserved Volume generated distinct magnetic induced frequency shift. resonance signals. 0367. In another set of calculations, the thickness of the 0372. The results of the experiment demonstrated that the mismatched disks in a dual-disk structure was varied to deter dual-disk magnetic resonance structures may be deactivated mine whether the mismatch in size could be compensated for by filling in the reserved Volume, and that the signal generated by variation in disk thickness. FIG.33 summarizes the results by the structures was dependent on the diffusion of fluid into of these calculations, showing the z-spectrum from FIG. 32 the reserved volume between the disks of a dual-disk mag for the dual-disk magnetic structure with identical disks netic resonance structure. (1.00), with one disk having a radius that was 85% of another (0.85), and with one disk that was 85% of the other, but the Example 10 Smaller disk is also thinner than the larger disk to compensate for the radius asymmetry. As shown in FIG. 33, variation in Effect of Non-Uniform Cylinder Wall Thickness on disk thickness may be used to partially compensate for dif Frequency Shifts of Hollow Cylinder MRS ference in disk radius. 0368. Another set of calculations varied the offset of the 0373 To assess the effect of variations in the thickness of centerlines of two identically-sized disks in a dual disk mag the walls of a single hollow cylinder magnetic resonance netic resonance structure by as much as 35% of the disk structure, the following simulatin was conducted. Rather than radius. FIG. 34 is a set of Z-spectra for various centerline dual-disk structures, the magnetic resonance structures were offsets in a direction perpendicular to the orientation of the hollow cylinders having increasingly non-uniformity in the applied magnetic field. FIG.35 is a set of z-spectra for various cylinder wall thickness. FIG. 19B is a series of simulated centerline offsets in a direction parallel to the orientation of Z-spectra Summarizing the results, showing diminished sig the applied magnetic field. In both FIG.34 and FIG.35, as the nal strength and peak broadening for the hollow cylinders centerlines of the disks are increasingly offset, there is a with non-uniform wall thickness. significant signal loss and peak broadening. 0374. The results of this experiment demonstrated that the 0369. Yet another set of calculations, the effects of cross signal strength and line width of a hollow cylinder magnetic wafer processing variations on the frequency-shift character resonance structure is relatively sensitive to variations from a istics of the dual-disk magnetic resonance structures were uniform wall thickness over the full length of hollow cylinder. assessed. In this set of calculations, the effects of up to 10% interparticle variation due to random variation in the manu Example 11 facturing process was estimated. FIG. 36 is a set of Z-spectra calculated assuming arrays of structures with varying degrees Effect of Cylinder Geometry Variation on Frequency of random variation in the manufacturing process. As the Shifts of Hollow Cylinder MRS variation of the manufacturing process increases past about 0375 To assess the effects of variations in the geometry of 1%, there was significant signal loss. a hollow cylinder magnetic resonance structure on the fre 0370. The results of this experiment demonstrated that quency shift characteristics of the structure, the following both systematic asymmetries, and random manufacturing simulations were conducted. Grids similar to those described errors of Sufficient magnitude may significantly impact the in Example 10, but for the use of hollow cylinder magnetic efficacy of the dual-disk magnetic resonance structure resonance structures rather than dual-disk structures, were through signal loss, which impacts the detectability of the measured using an indirect detection technique. Z-spectra structures, and peak broadening, which impacts the ability to were obtained using an 11.7 TMRI scanner for four different discriminate between structures with different geometries arrays of hollow cylinder geometries. All hollow cylinders used in multiplexed magnetic resonance visualization tech were constructed of nickel, an had an aspect ratio (length/ niques. This demonstrate the requirement of accurate micro diameter) of about 1.2. fabrication of these structures and precludes less accurate 0376 FIGS. 23A-23D show experimental Z-spectra chemical synthesis approaches. acquired from the four different arrays of hollow cylinder magnetic resonance structures. The hollow cylinders mea Example 9 sured in FIG. 23A had an outer diameter of about 2 um and a Deactivation of Dual-Disk Magnetic Resonance wall thickness of about 75 nm. The hollow cylinders mea Structures by Obstruction of Reserved Volume sured in FIG. 23B had an outer diameter of about 2 um and a wall thickness of about 150 nm. The hollow cylinders mea 0371 To demonstrate the effect of filling in the reserved sured in FIG. 23C had an outer diameter of about 850 nm and space between the disks of a dual-disk magnetic resonance a wall thickness of about 40 nm. The hollow cylinders mea structure, the following experiment was conducted. A grid of sured in FIG. 23D had an outer diameter of about 900 nm and dual-disk magnetic resonance structures Submerged in water a wall thickness of about 50 nm. US 2011/0014129 A1 Jan. 20, 2011 28 0377 Comparing the z-spectra of FIGS. 23A-23D, in one tube did not affect the spin-labeling in the other tube. increasing the wall thickness increased the magnitude of the As a result, each tube may be spin-tagged separately. frequency shift when comparing hollow cylinders of approxi 0382 To demonstrate the capability of the spin-labeling mately the same outer diameter. However, the magnitude of technique described above to perform perfusion imaging, the the frequency shift was dependent on a combination of all of flow tubes described above were spin-labeled using RF label the factors included in Equation 8 above. For example, the ing pulses spaced closely enough in time so as to appear magnitude of the frequency shifts for the smaller diameter continuous in subsequent MRI images. FIGS. 30 and 31 are cylinders, as shown in FIGS. 23C and 23D fall in between the MRI images taken from the left and right tubes, respectively. frequency shift magnitudes of the larger hollow cylinders, In FIG. 30, the flow labeled by a rapid series of RF pulses shown in FIGS. 23A and 23B. In all cases, the frequency appears as a solid continuous band having the same laminar shifts of the hollow cylinders fell within about 10% of the parabolic flow profile as in FIG. 28. In the more rapid flow frequency shifts predicted by Equation 8 above. shown in FIG. 31, a similar parabolic continuous band was 0378. The results of this experiment demonstrated the detected in the MRI image. magnitudes of frequency shifts induced by hollow cylinder 0383. The results of this experiment demonstrated that the magnetic resonance structures for a variety of geometries flow through tubes may be tagged in a local region by spin were in agreement with theoretical values predicted by Equa labeling the flow using a hollow cylinder magnetic resonance tion 8 above. By varying the geometries of the hollow cylin structure situated around the circumference of the tube and ders, a multitude of distinct signals may be generated for use excitatory RF pulses. The RF pulses may be discretely spaced in a multiplexed magnetic resonance visualization technique. in order to obtain information about finer features of the flow structure such as parabolic flow profile, or the RF pulses may Example 12 be closely spaced to produce an essentially continuous region of tagged flow for other flow visualizations such as perfusion Flow Tagging Using Hollow Cylindrical MRS imaging. 0379 To demonstrate the feasibility of flow tagging using Example 13 a hollow cylindrical magnetic resonance structure, the fol lowing experiment was conducted. Flow tagging is defined in Theoretical Single-Voxel Signal Due to Transverse this context as the process of frequency-shifting a plurality of Dephasing for Design of Solid Particulate MRS water protons and other NMR-susceptible nuclei in a moving 0384. To assess the effects of various factors such as the stream, rendering the frequency-shifted water protons and materials used to construct a solid particulate MRS, and the other NMR-susceptible nuclei in the flow detectable using a position of a solid particulate MRS within a voxel volume on magnetic resonance Scanner as the flow travels through a flow the contrast signal produced during magnetic resonance visu path. In this example, a large hollow cylinder magnetic reso alization, the following experiment was conducted. A theo nance structure was formed by wrapping a layer of nickel retical simulation of the magnetic resonance contrast was around the entire circumference of a region of a tube. Two performed using Solutions to the equations described below. tubes were tested in which one tube was wrapped to a thick 0385. The signal intensities of the solid particulate MRS ness of 50 um and the and the other tube to a thickness of were modeled theoretically assuming that the contrast signals about 100 um. originated from individual, micrometer-sized contrast par 0380 Water was passed through each of the two tubes at a ticles with high magnetic moments and that the signals were flow velocity of about 0.5 m/s and about 1 m/s respectively, as measured using high-resolution imaging. The calculations shown in FIG. 27. As the water passed through the nickel were simplified by assuming that the MRS fell within a static hollow cylinders wrapped around each tube, the water pro dephasing regime in which Act) t>>1, where A(i) was the tons in each pipe were periodically spin-labeled by a uniform local precession frequency due to the magnetic field of the magnetic field inside each hollow cylinder, together with MRS, and t, was the time to diffuse a distance equal to the exposure to a RF magnetic pulse at the offset Larmor fre size of the MRS. Ignoring k-space shifting effects, the time quency, defined by the above-mentioned uniform magnetic dependent modification to the magnetic resonance signal S field. The spin-labeled water protons produced a lower fid caused by the solid particulate MRS was proportional to an signal within the flow of water through the magnetic reso integral taken over all precessing spins within the Volume of nance imaging region downstream of each hollow cylinder interest as expressed in Eqn. (10): magnetic resonance structure. In this experiment, the RF magnetic pulses were applied to both tubes simultaneously. S(t)a?p(r)e-bred, (10) 0381 FIG. 28 is an MRI image formed after waterpassing 0386 where t was the time following excitation by an through the hollow cylinder was spin-labeled using three -e temporally separated RF pulses to the hollow cylinder of the initial U/2 electromagnetic pulse, r was the spin location left tube, which contained water flowing at about 0.5 m/s. The relative to the MRS, p was the spin density, and cp was the image of the left tube in FIG. 28 exhibited a characteristic additional accrued transverse phase due to the particle field in parabolic laminar flow profile from the three groups of spin the rotating frame. labeled flow. A similar result was obtained for the tube with a (0387. Since the MRS size was always far less than the flow velocity of about 1.0 m/s as shown in FIG.29. In FIG. 29, voxel size in this experiment, the signal produced by the MRS the three layers of spin-tagged flow are more spatially dis was dominated by spins from the far-field region of the MRS. persed along the direction of the flow due to the higher flow As a result, the magnetic field induced by the MRS was velocity. The distinct frequency shifts induced by the two modeled as a magnetic dipole independently of the shape of hollow cylinder thicknesses used in this experiment was dem the MRS. The MRS was assumed to be a sphere of radius onstrated by the observation that the spin-labeling of the flow determined by its net dipole moment p, and the magnetic US 2011/0014129 A1 Jan. 20, 2011 29 saturation of its constituent material. For a Bo-field aligned in 0391 Although the equations developed above assumed the z-direction, the z-component of the field produced by the spherical Voxels, the equations were adapted to cubic Voxels MRS when magnetized by Bo was B. p. (Lo?4t)(3 cos’ shapes by replacing the spherical Voxel radius R with an effective cubic “radius R according to the relation (4/3) 0-1)/r? for a magnetic permeability lo-4t 107 H/manda atR=8R, where R is the half-width of a cubic voxel. polar angle 0. For a ferromagnetic or Superparamagnetic par 0392 Theoretical voxel intensities induced by a 1-um ticle having a radiusa and saturation magnetic polarization.J. diameter spherical MRS particle with a J-1 T was estimated the dipole moment is p, (J/lo):47ta/3, resulting in an equa using the equations and methods described above. torial precession frequency of Aco-YJ/3 for the gyromagnetic 0393 FIG. 43 is a graph summarizing theoretical single Voxel signal intensities relative to background signal intensity ratio Y. due to transverse dephasing effects of a solid particulate MRS 0388 Neither Bo nor the magnetic susceptibility differ centered within a spherical VOXel having a diameter of 50-um, ence A affects the equatorial precession frequency, since 100-um, or 200-um. FIG. 44 is a similar graph Summarizing ferromagnetic and Superparamagnetic Substances are magne theoretical single-voxel signal intensities within cubic VOX tized to saturation by typical Bo fields. The normalized signal els. For both FIGS. 43 and 44, as the echo time TE increased, decay from Such a particle centered in a spherical voxel of the contrast increased until a saturated contrast level was radius R and of homogeneous spin density may then be reached. This saturation contrast was highest for the Smallest expressed: voxel size in both spherical and cubic voxels. 0394 The results of this experiment demonstrated that the solid particulate MRS produced suitable signal contrast for R a S(t) 3 2 2 : (11) gradient-echo MRI visualization, particularly at the smaller S(0) 2(R3 a? ex-Art s (3cos 6 - 1). r. sinédrale voxel size of 50-100 um. Example 14 0389. Although the high magnetic moments typical of the MRS to be modeled precluded immediate expansion of the Theoretical Single-Voxel Signals with and without integrand in Eqn. (11), simplification was still possible if the Image Distortion Correction ratio of voxel to particle radius (AG))(t)(a/R)) was on the 0395. To assess the effects of image distortion on the con order of unity or less. For millisecond timescales and trast signal produced during magnetic resonance visualiza micrometer-sized ferromagnetic particles, this condition was tion, the following simulation was conducted. Although the fulfilled at magnetic resonance resolutions of about one hun image darkening produced by a contrast particle is typically dred micrometers or larger. By integrating first, and then dominated by T transverse dephasing, for magnetic reso simplifying the resultant functions of Aco(t) and Aco(t)(a/R) nance conditions such as high-resolution imaging and short through asymptotic and power series expansions, respec echo times, geometric image distortion may also appreciably tively, the signal magnitude was approximated to second modify the contrast signal intensity. The image distortions order in Aco(t)(a/R): result from the Superposition of the contrast particle's field onto the read magnetic field gradient, resulting in local hypointense and hyperintense contrast signal regions near the S(t) a a (12) contrast particle. If the hypointense and hyperintense contrast so, s 1 -c. Aco. 1 + c, Aco. 1 + higher order terms signal regions fall within the same Voxel, conservation of spin number ensures that the spatial variations in apparent spin density cancel out within the Voxel, resulting in negligible in which: image distortion effects. However, if hypointense and hyper intense contrast signal regions fall across two or more Voxels, as may be the case for high-resolution magnetic resonance visualization, then image distortion may appreciatively C -3V32 and3C c.C 2 =-i 5 -to il-Met I - -V3 Y. - . change the Voxel signal intensities. 0396 To approximate the length scale of the image distor tion effects, higher-order slice selection effects were ignored 0390 The quadratic term in Eqn. (12) represented the and 3D imaging with a read gradient of strength G in the onset of signal saturation due to finite Voxel size. Despite the x-direction was assumed. The field of the solid particulate limited expansion of terms, Eqn. (12) accurately approxi MRS during read-out was therefore G+B if the Bo offset is mated the solution for the integral in Eqn. (11) for initial ignored. Spins located at a position X map to an apparent signal decay and Saturation. Comparing the linear and qua position (X+BAG), and hyperintense and hypointense signal dratic terms in Eqn. (12) estimated the dephasing period (t) maxima result when GBAGX takes on a minimum or maxi required to appreciably saturate out the Voxel signal. mum value, respectively. Simplifying the analysis by setting Although the asymptotic nature of signal saturation made the y-Z 0, setting GB/6x0 gave X-(Ja/G)' and an associ definition of the dephasing period somewhat arbitrary, as a ated hypointense signal maximum mapped a distance daway first measure the point at which S(t) became stationary, as from the solid particulate MRS given by: approximated by Eqn (12) was used to estimate the dephasing period: (14) d = 4,W Ja ()'s3 ( 3 (13) 0397. With a compensating hyperintense signal maxima similarly displaced away from the solid particulate MRS, the US 2011/0014129 A1 Jan. 20, 2011 30 ratio of d to the voxel size predicted whether image distortion Example 15 significantly modified the initial signal magnitude. Although distortion was also affected by whether Bo was parallel or Image Simulation with Solid Particulate MRS perpendicular to the image plane, the distance d was 0400. To compare the effects of magnetic materials used to unchanged, thus the overall distortion sizes did not depend construct solid particulate MRS, the echo times and the voxel strongly on the direction of Bo. Even with high magnetic resolution on the contrast signal generated by Solid particu moment MRS, image distortion was significant only for high late MRS, the following experiment was conducted. Simu resolution imaging becaused scaled as the fourth root of the lated magnetic resonance images were modeled using the methods described in Examples 14 and 15 using three differ magnetic dipole moment in Eqn. (14). However, for modeling ent contrast particle geometries and compositions. As a ref the detection of single particles, high-resolution imaging was erence, a commercially-available micrometer-sized iron taken into consideration. For example, Substituting a 1-um oxide particle (MPIO. Bangs Laboratories) was modeled as a diameter, J-1 T particle and a typical high-resolution imag 1.63-um diameter beads composed of 42.5% magnetite by ing gradient G of a few Gauss/cm Eqn. (14) indicated that weight and having a total magnetite content of 1.5 pg. Rep image distortion may contribute to signal strength at Voxel resentative microfabricated disks were modeled as disks of sizes of about one hundred micrometers or less. pure nickel and iron having a diameter of 2-um and a thick 0398. For solid particulate MRS with high magnetic ness of 300-nm. Both microfabricated disks were surrounded moments and high-resolution imaging, therefore, distortion by 50 to 100-nm thick shells. All particles had roughly com may dominate the signal in the first few milliseconds follow parable total Volumes. The magnetite, nickel, and iron mate ing the initial excitation, after which the signal magnitude rials had J. values of approximately 0.5 T. 0.6 T, and 2.2 T. may be dominated by dephasing effects described by Eqn. respectively. As a result, the respective magnetic dipole (11) above, which was assumed valid until the voxel signal moments of these particles were approximately 0.1x10' started to appreciably saturate around the Voxel signal Satu A m, 0.45x10'A'm, and 1.65x10' A'm. All particles ration time t, defined by Eqn. (13). In order to capture were assumed to be centered within the image voxel. The dephasing, distortion, and Saturation effects simultaneously, characteristics of the three contrast agent particles are sum the gradient-echo imaging of individual magnetic particles of marized in Table 1 below: TABLE 1. Contrast Agent Material Properties Magnetic Coating Js of Magnetic Type of material thickness magnetic dipole Contrast Diameter Thickness purity (% and material nonent Agent (Lm) (nm) wt) material (T) (A. m.) MPIO 1.63 42.5 — O.S O.1O. 1012 sphere nickel 2 3OO 1OO Gold, 50-100 nm. O6 O45. 10-12 disk iron disk 2 3OO 100 Gold, 50-100 nm. 2.2 165. 1012 various moments and at various image resolutions and echo 04.01 FIG. 46A-46D summarizes the results of the mag times were simulated. The simulation model tracked the netic resonance image simulations. The imaging simulations phases of a Volume of spins precessing in the magnetic field for the MPIO, nickel, and iron particles captured image dark Surrounding a magnetic dipole, with intravoxel dephasing ening over several voxels rather than in only the central voxel captured through a grid spacing many times Smaller than the as previously predicted in Examples 13 and 14. For each simulated Voxel size. The apparent image location of each particle, the images show theoretical (noise-free) pixelized spin was determined by the net magnetic field at that spin's gradient-echo signals for various echo times from individual real location, given by the sum of the perturbing dipole field particles at 50 and at 100 micrometer (cubic) isotropic reso and a simulated readout gradient. lution and for Bo oriented in-plane and perpendicular to the 0399 FIG. 45 is a graph summarizing theoretical single imaging plane. As expected, image distortion modifies the Voxel signal strength from a solid particulate MRS as a func images of the nickel and iron particle signals initially, before tion of echo time TE calculated for cubic voxel sizes of dephasing effects begin to dominate at higher echo times. 50-um, 100-um, and 200-um. The solid lines summarize the 0402. The results of this experiment demonstrated that the Solutions obtained for theoretical Voxel signal strengths for micromachined solid particulate MRS generated higher con numerical simulations that included both transverse dephas trast signals than a similarly-sized existing MPIO contrast ing and image distortion, and the dashed lines Summarize the particle. results obtained for transverse dephasing effects only. At the larger Voxel size of 200-um, image distortion had a negligible Example 16 effect on signal strength. However, as the Voxel size and/or the Effect of Off-Center Placement of Microfabricated echo time TE decreased, image distortion effects became Solid Particulate MRS in Voxels. During Gradient increasingly pronounced. For example image distortion Echo MRI on Signal Strength effects reduced the voxel signal strength by about 50% as the echo time approached Zero for the 50-um voxel size. The 0403. To assess the effects of the location of a contrast results demonstrate that image distortion must be taken in particle within a voxel during echo-gradient MRI on the acCOunt. image darkening produced by the contrast particle, the fol US 2011/0014129 A1 Jan. 20, 2011 lowing simulation was conducted. Although fractional Voxel images taken at a 100-um isotropic resolution. The left col offsets have little impact at those resolutions where image umn (FIGS. 48A and 48D) are images of the MPIO contrast darkening extends over many Voxels such as in higher reso particles, the center column (FIGS. 48B and 48E) are images lution magnetic resonance visualization performed using of the nickel disks, and the right column (FIGS. 48C and 48F) relatively very Small Voxel sizes, signal hypointensities drop are images of the iron disks, all described previously in Substantially due to increased signal dilution arising from Example 15. partial Volume effects in lower resolution magnetic resonance 0410 AS expected, higher magnetic moment particles visualization. For particles aligned in the middle of an imag caused more pronounced image darkenings. Localized ing slice, therefore, identical particles may appear differently hypointense regions in all images were assumed to be prima depending on their lateral registration with regard to their rily due to single particles due to the low particle concentra respective imaging Voxels. tions used and by the good agreement, at least for the micro 0404 Simulated magnetic resonance images were calcu fabricated particles, between the calculated and lated for the three different particles described in Example 15 experimentally measured signal intensities. in a similar manner. However, in addition to generating an 0411 To quantitatively compare the calculated and magnetic resonance image assuming that the particle was experimentally measured signal intensities, all 100-microme centered within its imaging Voxel, three additional locations ter resolution image slices including those shown in FIG. 48 of the particle within the voxel were calculated. The four were analyzed using image analysis Software that automati locations are illustrated in FIG. 47 and marked as A (cen cally selected and recorded the pixel intensity of all localized tered), B (centered on right edge of voxel), C (centered on top dark regions in each image. The data were collected into edge of Voxel), and D (corner of Voxel). All simulated mag histograms of normalized signal intensities (SCTE)/S(O)) netic resonance images were calculated assuming 100-um approximated from the images by taking the ratio of signal voxel resolution and a 10 ms echo time. intensity of the darkest voxels in each darkened region to the 04.05 FIG.47 summarizes the magnetic resonance images signal intensity averaged from a particle-free region of the calculated for the three different contrast agents described in sample image. For comparison, the experimental intensity Example 15. For each of the contrast agents, position-depen distributions integrated the signal intensity over variations in dent signal dilution was observed. Signal hypointensity particle magnetic moment, particle registration with respect decreased as dephasing effects were averaged over an to lateral Voxel position and image slice height, and back increasing number of Voxels, and decreased most severely ground noise. when the particle was located at the corner of a voxel, where the dephasing effects may be averaged over as many as eight 0412 FIGS. 49A-49C are histograms summarizing the Voxels in three-dimensional imaging. The position-depen simulated and experimentally measured signal intensities of dent signal dilution rendered the MPIO contrast particle the iron disks, nickel disks, and MPIO contrast particles, invisible when the particle was located at a voxel corner respectively. For all contrast particles, the simulated and the (position D in FIG. 47). experimental histograms showed hypointensity distributions 0406 Overall signal hypointensity ranges were approxi that were non-Gaussian and too broad to be explained by mated using the simulated magnetic resonance images background noise alone. Instead, the histograms indicated because the echo time used was selected Such that image that the contrast signals were dominated by subvoxel-level distortion and the Saturation of Surrounding Voxels was neg variation in the particle location. ligible. For example, in moving from a central point (A) to a 0413. The results of this experiment indicated that the corner point (D), signal hypointensity drops nearly four-fold experimentally measured magnetic resonance visualization in two-dimensional imaging. Similarly, in three dimensional signal intensities of the contrast particles were in good agree imaging an offset from Voxel center to corner reduces signal ment with theoretically predicted signal intensity calcula hypointensity as much as eight-fold. tions for the solid particulate MRS. For comparison, the 0407. The results of this experiment demonstrated that the experimental intensity distributions, which integrated the sig strength of the signal of a contrast particle during magnetic nal intensity over variations in particle magnetic moment, resonance visualization is sensitive to the position of the particle registration with respect to lateral VOXel position and contrast particle within the imaging Voxel. Further, in order to image slice height, and background noise, were compared to enhance the visibility of the contrast particle at arbitrary theoretical (background noise-free) calculations. placement within the Voxels during magnetic resonance visu alization, high magnetic moment contrast particles Such as Example 18 the nickel or iron disks may be preferred. Determination of Minimum Particle Moments to Example 17 Ensure Visibility of Solid Particulate MRS Comparison of Experimental MRI Images of Solid Particulate MRS VS. MPIO 0414. To determine the minimum particle magnetic moments necessary to assure the visibility of the solid par 0408. To compare the visibility of solid particulate MRS ticulate MRS during echo-gradient MRI, the following simu contrastagents to existing iron oxide micro-particles (MPIO), lation was conducted. Contrast signal intensities were calcu the following experiment was conducted. Contrast particles lated for a spherical particle with a magnetic moment ranging similar to those described in Example 15 were suspended in from about 10' A'm to about 10' A musing the methods three separate agarose samples and Subjected to T described in Example 15. At each magnetic moment, signal weighted, 12 ms TE, gradient-echo MRI at 50-um and 100 intensities were calculated for an ensemble of particles that um isotropic resolution. were positioned randomly with respect to the imaging Voxel 04.09 FIGS. 48A-48F are representative MRI images locations. Simulated magnetic resonance visualization signal obtained for the three different contrast particles. The top row intensities were calculated assuming an isotropic resolution of images (FIGS. 48A-48C) is images taken at a 50-um iso of 50-um and 100-um, and echo times of 5 ms, 10 ms, and 20 tropic resolution, and the bottom row (FIGS. 48D-48F) are S. US 2011/0014129 A1 Jan. 20, 2011 32 0415 FIGS. 50A-50F are graphs summarizing the calcu 2. The magnetic resonance contrast agent of claim 1, lated image intensities for all conditions described above. wherein each disk of the plurality of disks has a magnetic Empirical MPIO contrast signal data are superimposed on moment from about 10' A'm to about 10'' A. m. FIGS. 50C and 50D for comparison. The lower boundaries of 3. The magnetic resonance visualization contrast agent of the relative signal intensities are all greater than Zero, but claim 1, wherein each disk of the plurality of disks vary in size must be greater than background noise signals to be visual. by less than about 5% of the average size of the plurality of Contrast particles having magnetic moments above the 10' disks. Am threshold are predicted to be visible above relatively 4. The magnetic resonance visualization contrast agent of low background noise for all conditions examined. This mag claim 3, wherein each disk of the plurality of disks vary in netic moment threshold is slightly lowerfor higher echo times magnetic moment by less than about 5% of the average mag and/or higher image resolutions. netic moment of the plurality of disks. 0416) The results of this experiment predicted that con 5. The magnetic resonance visualization contrast agent of trast particles having a magnetic moment of at least 10' claim 1, wherein the magnetic material comprises a ferro Amare visible above low background noise overavariety of magnetic, paramagnetic, Superparamagnetic, magnetic alloy, magnetic resonance visualization conditions. magnetic compound, or any combination thereof. 6. The magnetic resonance visualization contrast agent of Example 19 claim 5, wherein the magnetic resonance contrast agent fur ther comprises a coating selected from an oxidation barrier, a Super-Resolution Tracking Using Solid Particulate corrosion barrier, a mechanical strengthening layer, a spatial MRS buffer coating to inhibit the clumping of magnetic resonance 0417. To demonstrate the potential use of solid particulate contrast agents, a non-toxic coating, a biologically inert coat MRS in super-resolution tracking, in which the sub-voxel ing, a coating to facilitate common bioconjugation protocols, location of a particle may be determined, the following a cell-specific antibody or ligand coating, and combinations experiment was conducted. Solid particulate MRS that thereof. included iron disks coated in gold, similar to those described 7. The magnetic resonance visualization contrast agent of in Example 15 were Suspended in agarose and Subjected to claim 1, wherein the magnetic resonance visualization con echo-gradient MRI as described in Example 17. trastagent is a disk shapehaving a disk diameterranging from 0418. A representative MRI image is shown in FIG.51. In about 0.1 um to about 10 Lum and a disk thickness ranging FIG. 51, individual solid particulate MRS were detected from about 0.1 um to about 10 Lum. within the image. In addition, due to the consistent size and 8. A method ofusing two or more MRS, wherein each MRS composition of the solid particulate MRS, the signal intensity induces a known NMR shift in a NMR-susceptible nucleus, of an individual solid particulate MRS could be compared to wherein each of the known NMR shifts and each of the the signal intensities of the other solid particulate MRS to corresponding resonance frequencies is different for each of determine the location of the particle within the imaging the two or more MRS, the method comprising: voxels. For example, the darkest voxels were assumed to (a) distributing the two or more MRS within a sample: indicate particles centered within the voxel, and the lighter or (b) exposing the two or more MRS within the sample to more dispersed particle signals were assumed to be off-cen excitatory electromagnetic pulses delivered at each of ter. The degree of lightening and/or signal dispersion were the two or more corresponding resonance frequencies: used to narrow down the location of the particle within the imaging Voxels using the information from the calculated (c) obtaining nuclear magnetic resonance data after each image intensities shown in FIG. 47. On FIG. 51, individual excitatory electromagnetic pulse; and particles are circled and labeled using a similar convention to (d) using the known NMR shifts to determine the identity that of FIG. 47: the letter Adenotes particles centered in the of each of the two or more MRS. imaging Voxel, B and C denote particles on the top or side 9. The method of claim 8, wherein each particular MRS is edges of a Voxel, respectively, and D denotes a particle on a targeted toward a particular tissue or cell. voxel corner. 10. The method of claim 9, wherein the MRS targeted to a 0419. The results of this experiment demonstrated that the particular cell is bound to the cell at a cell wall or cell mem uniform composition and size of the solid particulate MRS brane. enabled these contrast particles to be used for super-resolu 11. The method of claim 9, wherein the MRS targeted tion tracking. toward a particular cell is situated anywhere within the cell. 0420. The invention has been described in detail with 12. The method of claim 8, wherein each different NMR respect to various embodiments, and it will now be apparent shift is assigned a different color on a color scale. from the foregoing to those skilled in the art that changes and 13. A method of non-invasively monitoring at least one modifications may be made without departing from the inven characteristic of blood flow through a stent device situated tion in its broader aspects, and the invention, therefore, as within a blood vessel of a living Subject, comprising: defined in the claims is intended to coverall Such changes and modifications as fall within the true concept of the invention. a. providing a stent device comprising an MRS, wherein the MRS induces a known NMR shiftina NMR-suscep tible nucleus; 1. A magnetic resonance contrast agent consisting essen b. situating the stent device within the blood vessel of the tially of a plurality of disks of uniform size and magnetic living Subject; moment, wherein the disks consist essentially of a single c. exposing the stent device to at least one or more excita magnetic material. tory electromagnetic pulses delivered at the correspond US 2011/0014129 A1 Jan. 20, 2011 ing resonance frequency to create a Volume of spin 15. The method of claim 13, wherein the characteristic of labeled blood molecules; blood flow is compared to a baseline characteristic of blood d. obtain nuclear magnetic resonance data of a Volume of flow to determine the presence or absence of occlusions blood flowing downstream of the stent device; and, within the stent device. e. analyzing the magnetic resonance image data to locate 16. The method of claim 13, wherein the NMR shift of the the volume of spin-labeled blood molecules using the spin-labeled blood molecules is compared to a baseline NMR known NMR shift. shift to determine if any deformation in the shape of the stent 14. The method of claim 13, wherein the at least one device has occurred. characteristic of blood flow comprises mass flow rate, volume flow rate, and flow speed.