© 2012 Nature America, Inc. All rights reserved. need to be made to a laser-scanning microscope to enable the measurements. an overview of the state of the art and the physical background of more structural information on the assembly of collagen in tissues than is possible by other microscopy techniques. We first providechanges that occur in diseases such as cancer, fibrosis and connective tissue disorders. We discuss how of tissues. Because of its underlying physical origin, it is highly sensitive to the collagen fibril/fiber structure, and, importantly,S to Published online 8 March 2012; doi:10.1038/nprot.2012.009 to P.J.C. ([email protected]). Utrecht, The Netherlands (O.N.); US National Institutes Health,of Heart, Lung and Blood Institute, Bethesda, Maryland, USA (S.P.). Correspondence should be addressed 1 Xiyi Chen quantitative analysis of collagen fibrillar structure Second harmonic generation microscopy for 654 second The anti coherent and processes Raman stimulated and (THG) generation harmonic third tion, tion; and the third two term describes second term describes SHG, sum and difference frequency genera the light; of reflection and scattering absorption, linear describes susceptibility tensor and where can light with be generally interacting expressed as response. Nonlinear optical Physical and chemical background the technique and to refs. referencesto directed is component parts andthe perform from thesetup required our calibrations.implement to Therequired reader be would weeks 2–4 that estimate we experience, user’s of a on level tocol.Depending students working in this field should be able to implement this pro clearing optical of mechanism the investigations of ovarian cancer techniques the detail developed in in our laboratory, which we describe have previously used in we our end, this To SHG microscope. the in information structural collagen obtaining for used tions with representative references is given in is not meant to be comprehensive, an overview currentof applica contrast mechanisms observed in SHG microscopy theoretical analyses have been developed to physically interpret the diseases of range wide a diagnosing for medicine,andprovided hasbiology itquantitativein and metrics detail supra­ molecularthe visualizing assembly for tool of collagen powerful a in as tissues emerged at has an copy unprecedented level of Over the past decade, the nonlinear optical method of SHG micros­ I instrument from its component parts will require 2–4 weeks, depending on the level of the user capabilities and limitations of the different experimental configurations. We estimate that the setup and calibration of the Department of Biomedica l of UniversityEngineering, Department of Wisconsin NTRO protocol econd-harmonic generation ( In this article, we focus on the instrumentation and methods methods and instrumentation the on focus we article, this In

| 4 and and 4 VOL.7 NO.4VOL.7 1– P 5 D . This development has enabled and enhanced basic research is the induced polarization, UCT 5 for earlier descriptions of the most salient aspects of of aspects salient most the of descriptions earlier for 5 - 1 order symmetry of SHG imposes severe restrictions restrictions severe imposes SHG of symmetry order I , Oleg Nadiarynkh ON | E P 2012 + = c c c Box ) ( | 1 1

natureprotocols E 1–3 for current reviews of SHG imaging, imaging, SHG of reviews current for 1–3 is the electric field vector 1 for a glossary of terms. of for a glossary The total polarization for a material for material a total polarization The - Stokes Raman scattering (CARS). (CARS). scattering Raman Stokes S ) ( 1 HG) microscopy has emerged as a powerful modality for imaging fibrillar collagen in a diverse range 2 2 0 , connective tissue disorders E E 2 1 9 , 2 . It is our intent that graduate graduate that intent .our is It χ + + , Plotnikov Sergey ( - n and three ) is the 6–2 ) ( 3 3 5 . Concurrently,.several Table n th th order nonlinear  - photon absorp 3 26–2 0 . The first term 1 - 8 . Madison, Madison, Wisconsin, USA. . Although it 1 , 2 & Paul J Campagnola 1 1 and (1) ­ ­ ­ ­

S HG microscopy, and then describe the optical modifications that sented a more general model of SHG from tissues by considering considering by tissues from SHG of model general more a sented observed in cornea and sclera sues such as tendon successfully the SHG described emission pattern from regular tis have size fibril on based treatments example, For subject. the on the literature in recent years, and there remains some controversy these directional components has received attention in important respect (with to the laser direction of propagation). The nature of components propagating backward and forward comprising tern in tissue; see below), meaning that there is a specific emission pat SHG emission directionality. III collagen type also does not produce imageable SHG contrast imaging for signals SHG sufficient produce not does epithelium from the underlying stroma), is not fibrillar a component primary the of basement membrane the (separating acto type I and II collagen are requirements these meet that proteins primary nonzero.The be aligned within the focal volume of the microscope so that must these SHG, efficient for further,and, moment, dipole nent average. have must perma Thus, a orientational harmonophores where the by related then are properties formula: following bulk and molecular The dipole moment permanent the in terms of mechanism.is defined parameter This i.e., hyperpolarizability, first the ment.However, molecular the χ of scale size the on noncentrosymmetric be must environment the imaged, be can as that assembly their and harmonophores the on (2) To help resolve results, disparate the some seemingly of we pre λ , is a bulk property and is the quantity measured in an experi an in measured quantity the is and property ,bulk a is SHG - myosincomplexes N ; otherwise, the signal will vanish. The susceptibility tensor, s is the density of molecules and molecules is the the brackets of density denote their C rucial aspects for biomedical applications are the 2 Present Physics,of addresses: Department University Utrecht,of 1 3 4 3 , but they have failed to account for the results (both form aligned fibers) and myosin within 5,3 b c 1 ) ( . In contrast, type IV collagen, which is is collagen,which IV type .contrast, In 2 E d ′ s experience. ) ( 2 < = 2 SHG is coherent (or quasi 0 - . level property of the nonlinearity, the of property level N = β s , forms the basis of the contrast contrast the , of basis the forms b S HG can be used to obtain E >

3 2 in vivo . Similarly,. - S coherent HG χ and (2) (3) (2) is 3 3 ­ ­ ­ ­ ­ .

© 2012 Nature America, Inc. All rights reserved. T best described best as described quasi backward matching gives rise to a corresponding distribution of forward results in a distribution of nonzero tissues biological real in dispersion and randomness inherent the Although collagen has been described as a nematic liquid crystal and (KDP) phate for (e.g.,SHG from dihydrogen potassium crystals uniaxial phos ward directed and co and incident photons, respectively, the SHG emission is 100% for mismatch, and i.e., matching, phase fect to SHG and intensity directionality phase relaxed a Theory Model tissues Atherosclerosis and disorders Connective tissues Fibrosis Cancer A b pplication le le 1 1 |

- Representative example applications of SHG. emitted components, and as a result SHG in tissues is is tissues in SHG result a as and components, emitted - matching conditions and showed how these relate relate these how showed and conditions matching k 2 ω and β

- - barium borate (BBO)) and from interfaces. interfaces. from and (BBO)) borate barium propagates with the laser. This situation holds - k coherent. The phase mismatch also affects Skin Skin damage Cornea Sjogren’s syndrome imperfecta Osteogenesis Kidney Liver Skin Ovary Breast S ω ubspecialty are the wave vectors (2 ∆ k

=

k 2 ω

− ∆ 2

2 7 k . For the per case limiting of k

values. This imperfect phase ω

=

0, where where 0, R epresentative refs. π / ∆ λ k ) ) for the SHG is the phase phase the is 28 27 26 25 19 16 15 17 20 18 12 11 13 14 24 23 22 21 10 9 8 7 6 - and 3 5 ­ ­ ­ ,

SHG SHG delineates normal and diseased states in several tissue types SHG results agree with standard pathology SHG results agree with standard pathology SHG can delineate tumor boundaries in different types of skin cancer SHG shows an increase in collagen fibril/fiber organization SHG can delineate cancers of different stages Key conclusions/observations SHG SHG emission properties in tissues Theoretical treatments have been developed to understand the fibrillar Self-assembled gels can be imaged by SHG SHG shows that collagen plaques intermingle with elastin SHG uniquely shows changes in collagen assembly upon thermal damage SHG can delineate the stroma from other corneal components SHG shows disorganized collagen in this disease showed that the alignment must also be considered to properly properly to considered be also must alignment the that showed size fibril on based were that treatments previous ≥ values will be relatively less forward directed (although (although directed forward less relatively be will values larger with structures more random and/or from smaller sion emis the whereas SHG, forward predominantly to rise give will structures that are ordered on the size of Specifically, emission. SHG backward direct with associated are how larger havemodel, we our described and,by using assembly, fibril/fiber the of regularity the on depends ratio this is the coherence length and sin( as scales intensity,which SHG relative the that that the SHG becomes less efficient for larger values of 1) We have denoted the emission directionality 2 7 . For regular tissues, such as tendon, our theory agrees with with agrees theory our tendon, as such tissues, regular For. natureprotocols m is an integer. By this relation, we see λ SHG

| VOL.7 NO.4VOL.7 in the axial direction F m SHG protocol ∆ 4,3 kL / B 4 , but we also also we but , /2), where where /2), SHG | ∆ 2012 ∆ F , wherein k SHG k . values values / | B

655 SHG ∆ L k ­

© 2012 Nature America, Inc. All rights reserved. 656 at photons these emitted directionality ( initially the convolutionof a comprise will (F/B) signal SHG the and fibers fibrils organized randomly more smaller, have tissues diseased the microscopy, electron by intensity SHG lower respondingly ∆ by weregreater tissues characterized imperfecta osteogenesis that differentiate normal and diseased tissues. For example, we showedto exploited be can mismatch phase of degree The photons. ted arising from forward and backward scattering of the initially emit and F/B,components the emitted directionality which consists of as define we which experiment, an in measured quantity the not predict protocol k Box 1 Secondary filtereffect:the loss of signal inthe sampleasa result of scattering and/or absorption. linear tocircular polarization. Quarter-wave plate, or Primary filtereffect:the loss of laser excitation inthe sampleasa result of scattering and/or absorption. can beusedtoverifypolarization states. Polarizing beamsplitting cube:thisoptic isrelated toaGlan-typepolarizer, and ittransmits and reflects orthogonal polarizations and Pockels cell:anelectro-optic modulator incombination withaGlan-laserpolarizer;itisusedtocontrol powerand/or repetition rate. tions from the oppositedirection isrejected withpolarizers. Optical isolator:thisdevice functions as anoptical diode inwhich light isonlytransmitted inone direction, and light of allpolariza microscope, thiscorresponds toapixel sizeof halfthe diffraction limit. Nyquist criterion: thisprinciple statesthattoproperly identify asignal, itmust bedigitally sampledatleasttwice itsfrequency. Ina ­structural information onthe structure of the harmonophore. average of the first hyperpolarizability, Second-order nonlinear susceptibility, and apinhole. and directed onto the detector. Unlike confocal detection, the desired signal does not passbackthrough the galvoscanning mirrors Nondescanned detection: inthisoptical geometry, the signal inthe epidirection isisolatedwithadichroic mirror inthe infinityspace polarization direction. Half-wave plate, or broadening of the pulse in an optical material, which arises becauseGroup the velocityred and dispersion blue components (GVD): GVD have is differentthe dispersion refractive in velocities indices. of the frequency componentsbetween. The polarizerisrotated toselectthe transmission of adesired linear polarization.of a short laser pulse. It results in temporal Glan-laser polarizer(GLP):Thistypeof polarizerisconstructed from twoback-to-backbirefringent prisms withanairspace in First hyperpolarizability, F ­convolution of the emittedratio, which wedenote as F/B of SHG:wedefine thisquantity asthe measured ratio of forward and backward SHGcomponents inthe microscope. It isa field). Inamicroscope, these are used for polarization and powercontrol. Electro-optic modulator, orEOM: adevice using the electro-optic effect(i.e., the linear rotation of light inresponse toanelectric incident photons, respectively. where the coherence length, Coherence: for SHG,thispertains tothe phase(temporal and spatial) relationship betweenthe laserexcitation and the SHsignal, differing velocities. Birefringent tissues:such tissuesdisplaybirefringence (ordouble refraction) and decompose light into twodistinct rays of these canbeusedfor powercontrol and spectral selection. Acousto-optic modulator, orAOM: anAOM orBragg cellusessound wavestocreate agrating and diffract light. Inamicroscope, In a tissue imaging experiment, the measured directionality of of directionality measured the experiment, imaging tissue a In

aus hn h crepnig oml ise, n b cor by and tissues, normal corresponding the than values SHG

| VOL.7 NO.4VOL.7 / B SHG F SHG : wedefine thisasthe SHGcreation ratio (i.e., the initially emitteddirectionality of the SHGbefore scattering). | / B Glossary SHG | 2012 in less in λ SHG λ . The scattering behavior is governed by the the by governed is behavior scattering . The /2 plate:thisoptic retards the polarization of light byahalf-wavelength, or180°,and thereby changes the linear |

F natureprotocols λ - SHG organized tissues organized /4 plate:thisoptic retards the polarization of light byaquarter wavelength and thereby convertslight from β / : the molecular property thatgoverns the macroscopic SHGresponse. B SHG L c , isgivenby2 ) and the subsequent scattering of 2 7 . Accordingly, as confirmed confirmed as Accordingly, . 3 6 . χ β (2) . It isameasure of the SHGefficiency, and the 27-matrix-element tensor alsocontains : thisisthe bulkormacroscopic property measured inanexperiment and isthe 1 1 . We stress that this is is this . We that stress π / ∆ k , where F SHG / ∆ B k SHG

= , and the resulting scattering inthe tissue.

k 2 ­ ­ ω

−

2 ing type I collagen (or Col ing I),type which is the proteinmost abundant copy has been performed on tissues primarily or partially compris and size Collagen assembly scales. SHG the via imaging backward channel often will suffice. intensities, relative of comparison and lengths fiber of surement mea­ and visualization However, simple detection. for backward information the complete SHG microscope needs both forward and both of these arise from the fibril/fiber assembly, to fully exploit this information on and is related to tissue organization scattering of directionality the is latter the and direction, change a photon will distance propagate beforethe undergoing of a inverse scattering collisionthe and is it where density, of measure a is coefficient, scattering k ω , where k 2 ω and F SHG k ω / are the wavevectorsfor the SHGand B SHG µ s , and scattering anisotropy, ,scattering and and the scattering properties, and because 1 1 The majority of SHG micros of The majority . As the measured F/B contains

orientational g

. The former .former The

- ­ ­

© 2012 Nature America, Inc. All rights reserved. of differing matrix elements matrix differing of how normal and diseased tissues can be discriminated on the basis that contribute to the overall signal components chiral and achiral both delineated now have ments molecule collagen the in related to the pitch angle (~50 degrees) of the individual governing elements matrix nonvanishing the that showed previous analysis using polarization molecule the of length the ability, to hyperpolariz measurements showthe that scattering Rayleigh collagen.Forfromexample,Schanne SHG the There have been recent efforts to elucidate the molecular source of into fibers having diameters ~500 of nm to several micrometers assemble then which nm, 20–250 approximately of diameters of individual molecules then are self which the three triple a is Collagen body. the in fs, femtosecond; ps, picosecond; OPO, optical parametric oscillator. T except for the appropriate addition of filters modifications, additional any without setup scanning laser same the in implemented easily be can imaging TPEF and SHG ward intensity in weak fairly and nm) (~450–550 linked collagen has been observed, this emission spectrum is broad methods resolved directional and polarization through information tural struc more further,extract and,to labels, exogenous using from directly visualize protein assemblies without relying on inferences two points are important tabulated in mation capabilities with respect to collagen organization. The most with regard to microscope design and detection schemes and infor several linear and nonlinear optical microscopy methods, especially Here we provide a comparison the of salient features SHG of with other methods with Comparison cartilage, also efficiently produce SHG in predominantly found collagen, II type as such isoforms Other refs. (e.g., skin, bone, tendon), blood vessels and cornea (for reviews, see ovary,tissues (e.g., connective lung),liver, organs and nal kidney a OCT Two-photon SRS CARS THG SHG Method In general, the primary strength of SHG over the more common inter in SHG by imaged nowbeen has collagen Typefibrillar I b le le - 1–3). A montage representativeof tissues is given in photon excitation fluorescence (TPEF) is the ability to to ability the is (TPEF) fluorescence excitation photon 2 2 β , arose from coherent amplification of peptide bonds along 2 | 7

. Although two Comparison Comparison of nonlinear optical microscopy modalities. α Chemical, mostly C-H lipids Structural/assembly Structural/assembly P Structural Protein localization Chemical, mostly C-H lipids rimary information - chains chains are hydrogen - 3 photon excited autofluorescence of cross 9 . Other polarization .Other 3 8 . our consistentwas finding with This 4 2 . - - assembled covalently into fibrils Table helical molecule (~300 kD) in in kD) (~300 molecule helical 40,4 - resolved measurements, which 1 and have also begun to show 43–4 - bonded bonded to each other. The 2 5 . . 4 4 6 6 . . Weback that note - - resolved measure resolved Broadband fs Ti:sapphire ps/fs ps oPO, Cr:Forsterite fs Ti:sapphire L Klein used hyper used Klein asers

+

OPO

+

OPO α Figure Figure - χ helices helices (2) are 3 1 7 ­ ­ ­ ­ ­ ­ - - . .

optical sections show representative images of self-assembledFigure collagen 1 gel ( lipids lipids around collagen abundant C to relative bonds these of density low the of because proteins) other (and collagen in amides imaging for successful To the best of our knowledge, these Raman processes have not been and are thin specimens thus detected in the direction forward in lipids. These are coherent processes with ideal phase matching in specific bonds and have largely been used for imaging C (~333 nm) that is difficult to detect with conventional glass optics. edge of ~1,000 nm of Ti:sapphire excitationred the as detection, resultsTHG for needed inare wavelengthsexcitationTHG emission layerstissue between interfaces or surfaces tissue as such occurs, index refractive in change large a which in imperfect phase matching but it also contains a backward component in the tissue directionbecause of forward the in is emission the of majority the wherein mouse dermis ( Reflected, Reflected, interferometric 4 Forward (laser detection) Primarily forward F/B tissue dependent F/B tissue dependent E mission direction π - CARS CARS and stimulated Raman scattering (SRS) probe chemically SHG, as properties coherence same the of some shares THG H stretches. However, they can be powerful tools for imaging imaging for tools powerful be can However, they stretches. H c a

| Montage of SHG imaging of collagen tissues. ( b ); mouse bone ( 1 5 . natureprotocols 4 7 c . However, THG is restricted to regions ); and human ovary ( Bulk Bulk tissues Bulk tissues Bulk tissues Bulk tissues Interfaces tissue Bulk tissues to collagen A pplicability d b

| 4

VOL.7 NO.4VOL.7 8 . We note that longer .Wethat note d ). Scale bar, 30 a – protocol d ) The single Good Limited Limited Limited Limited Excellent specificity C ollagen - | H stretches 2012 µ m.

|

49,5 657 a ); 0 .

© 2012 Nature America, Inc. All rights reserved. established and consists of fibrillar collagen (e.g., types I and II) II) and I types (e.g., collagen fibrillar of consists been and established has SHG by imaged be can that harmonophores of scape land The proteins. structural of number small a to applicability limited its is microscopy SHG of limitation largest the Perhaps Limitations about 10–20 is typically OCT detection is limited by the bandwidth of the light source and mation polarization–resolved approaches OCT have been SHG, used to to extract more detailed analogy assembly In infor density. tissue to collagen and also arises from changes in refractive index and/or depths shallow very at or tissues) in (e.g.,refractive index specimens gels or either on the surface of microscopy has no molecular specificity but is sensitive to changes also been used to examine collagen assembly. Reflectance confocal have coherence microscopy (OCT) optical tomography focal and 658 ficient we have used optical clearing to greatly reduce the scattering coef tissues birefringent in signal SHG the excitation and the laser both of state polarization the toscramble shown that one or two scattering lengths (~20–50 mation are limited to fairly superficial regions. Specifically, we have scheme. collection depth for true floor noise the above well still was signal forward the whereas detected, not were photons backward which tissue in micrometers) muscle hundred (several skeletal thick imaging when effect this observed We microscope. the within elsewhere or objective the of radius collection the miss can direction backward the in scattering tiple mul undergo then and tissues in deep created are that photons signal geometry, this In acquisition. signal in factor limiting the be can filter secondary the tissues, scattering highly within SHG extent to utes a lesser contrib signal) SHG the of loss (i.e., effect filter secondary the of loss response, whereas (i.e., governs the intensity measured laser intensity) effect filter primary the of square the that mented docu have we approaches, simulation and experimental of tion combina a through detection, forward For schemes. detection note are that limits the for and actual different forward backward 2 ~1.5 an nm), (~1,200 excitation wavelength longer tissue). using By specific the of coefficient scattering the on thickness of (depending micrometers hundred few a about or lengths, ing scatter ~5–10 to limited are depths penetration achievable the linear optical techniques using near to penetrate and ability image highlythe scattering to tissues. related Similar are to other non modalities) optical nonlinear other by several laboratories potential membrane image to used been has approach this trast; membrane uses which SHG, probe–based levels contrast, imageable spindles,but mitotic and at muchyield lower here) and acto protocol - fold increase over Ti:sapphire wavelengths can be over achieved. wavelengths fold Weincrease Ti:sapphire Finally, the polarization analyses used to extract detailed infor detailed extract to used analyses Finally,polarization the are (which by SHG shared of microscopy the limitations Other con reflectance of techniques linear that mention we Finally,

| VOL.7 NO.4VOL.7 5,5 3 9 2 . Microtubule assemblies in live cells, such as centrosomes centrosomes as such cells,live in assemblies .Microtubule 4 9 52,5 . We stress that in this list we are not considering fluorescent , and we have demonstrated the retention of polarization polarization of retention the havedemonstrated we , and - myosin complexes skeletal within muscle (not discussed 3 . Unlike microscopy techniques, the axial resolution of of resolution .axial Unlike techniques,the microscopy in in vivo | 2012 applications, which by necessity will use this 10,11,2 | 55–5

natureprotocols µ 7 m. 9 2 . . For the case of detection of backward backward of detection .of case For the 9 . This has relevance for the achievable achievable the for .relevance has This 5 1 - . OCT is similarly nonspecific nonspecific similarly is OCT . IR excitation (700–1,000 nm), 58,5 9 .To limit,overcome this - staining dyes for con for dyes staining µ m) is sufficient - propagating propagating - to to ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­ ­

and co and Backman by presented was theory general a where architecture, from coefficient the arises scattering dependence the tral of tissue spec The scattering. with convolved be will which tissues, thick in dependence measured the for not and process, creation SHG tuning range. We stress that this holds only for the intensity the of lengths rigorously,More crucial. not thus is and the choice of excitation wavelength in terms of signal intensity about 10 nm at full width half fs,~100 of correspond towhich of a bandwidth widths pulse and 1–2 W MHz, averageof powers~80 of rates nm, repetition 1,000 ~700– of ranges tuning has that oscillator, Ti:sapphire pumped W) 5–18 nm; (532 Nd:YVO4 the is imaging SHG for laser used previous descriptions of the overall design SHG microscope is given in layout. optical and Laser design Experimental approach affords great into insight tissue structure clearing optical Although signatures through several hundred micrometers of tissue thickness L, lens; the optical components before the scan head and the detection pathways. Figure 2 that find we factors, these of basis the On ~30–40%. by broaden at 800 nm comparison,width will comparable temporal of pulses by ~10–20%. Thus, no external pulse compression is required. For increase only will width resulting the and range, wavelength this 100 for dispersion velocity nitrogen. the with laser purging cavity Finally, there is little group mately (approxi wavelengths longer at attainable is power lower hand, longer wavelengths result in better cell/tissue viability. On the other also avoid and autofluorescent scattering absorption bands decreased in tissues. of In addition, because depths penetration greater (~720 nm maximum) cross of band two autofluorescence the excited overlaps photon wavelength excitation SHG the if enhanced mirror Dichroic In the simplest interpretation, SHG is not a resonant process, resonant a not is SHG interpretation, simplest the In source excitation Femtosecond λ detection Forward SHG - 3

workers /2 and | >900 nm), and the range of ~940–970 nm also requires requires also nm ~940–970 of range the and nm), >900 0 but large changes are not expected across the Ti:sapphire Schematic of the optical layout of the SHG microscope, showing λ /2 λ /4 are half- and quarter-wave plates, respectively. 6 GLP 1 . We also note that the signal can be resonance resonance be can signal the that note We also . PM polarization Linear T 6 2 . Conversely, longer wavelengthsyield will SHG filter A schematic of the optical layout of the the layout optical the of of schematic A detection Backward SHG λ - in vivo in /2 Figure

fs pulses traversing the microscope in in microscope the traversing pulses fs λ

- /4 maximum maximum (FWHM). GLP polarization Elliptical is not practical at present, this this present, at practical not is 2 . We and others have provided L β PM decreases at longer wave longer at decreases T polarization Excitation Scanning 5,6 system 0 SHG . The most commonly filter or L - linked collagen collagen linked L Specimen ex ex vivo mirror Dichroic mirror Dichroic Condenser objective Focusing .

5 8 - ­ ­ ­ .

© 2012 Nature America, Inc. All rights reserved. to the entrance of ~2–3 the m),microscope (typically collimation used in our typically measurements. 900 the at width pulse the affect markedly not do 100 broaden will devices These nm. 300 a single device will not cover the entire Ti:sapphire tuning range of Unfortunately, the working bandwidths are typically ~100 nm, and provide ~40 dB of isolation, rejecting light typically of all devices polarization These states. locking. mode the annihilating and laser the entering from focus, of plane microscope the from especially also required to prevent reflections from upstream optical surfaces, An optical isolator (using a Faraday rotator; not shown in detectors. the background to co path microscope entire the can through Ti:sapphire for nm) (532 pump necessary residual as is detection, laser the following and Ti:sapphireviability performance. 900 We note that these conditions hold in the transverse plane rather rather plane transverse the in hold We conditions these that note fibers. oriented orthogonally visualizing against also discriminate will it contrary, the On PROCEDURE). the of 36 Step also see anis SHG the determine to exploited be can this and alignment, fiber and polarization laser of overlap the on dependent highly is sity packing fiber the on specifically data, structural tissue of extraction the affords imaging SHG in are considerably more expensive. vide similar performance and can be computer interfaced, but they of ~100 nm. Commercially available electro Zero in SHG dynamic intensity,of range which is more than sufficient. a factor ~100 of in power control, corresponding to a 10 range of control) motion relative you topolarization, GLP are the allowed the of angle the rotating fixing the output of the GLP to the desired linear and polarization polarizer (GLP; first set optics polarization of shown in a of combination a use we electro or optical either through Polarization AOMs. or filters ND either to relative achievable is range dynamic able) Moreover,vari purposes. continuously (and alignment greater a for choice superior a present and beam laser the deflect not do controlpower for microscope. methods Polarization the laser through the from alignment proper the retain to difficult also is it align; initially to difficult are devices power, these drive but (RF) optic modulator (AOM) can be used by varying the radio frequency filters offer limited dynamic range for power control. An acousto (ND) density power,neutral laser on intensity SHG the of ence Power and polarization control. resolution. sible and high tube lens, and will then lens appropriately fill transfer the back pupil aperture of medium the traversing upon beam, recollimated 1 a with achievable is 3 the on about 3 mm at the microscope entrance, and it may become clipped typical laser initial spot size of ~1 mm, the diameter will increase to is needed to correct for the ~1 mrad divergence of the laser. Given a The use of polarization analysis (both excitation (both analysis and emission) polarization use The of dichroic (SWP) pass wave short a that found Wehave Finally, because of the relatively long optical path from the laser - o nm nm excitation is a good compromise between depth,imaging - tropy (see ‘SHG polarization microscopy’ for more details; details; more for microscopy’ polarization ‘SHG (see tropy order wave plates provide accurate rotation over a bandwidth - - numerical o 4 to - mm - aperture (NA) objectives to yield the best pos - - based power adjustment can be achieved achieved be can adjustment power based diameter galvo mirrors. This collimation collimation This mirrors. galvo diameter - o 2 to λ λ /2 plate (either manually or through through or manually (either plate /2 /2 (half /2 - m focal length lens. The properly properly The lens. length focal m 1 Owing to the nonlinear depend - 0 . For example, the SHG inten SHG the example, For . optical control. In the former, the In control. optical - wave) plate and a Glan a and wave)plate - fs pulses at 700 nm, but nm, 700 at pulses fs - optic Pockels cells pro - propagate with with propagate - nm excitation excitation nm - free SHG SHG free Fig. Fig.

mirror mirror 2 - laser laser ). By 2 ) is - - ­ ­ ­ ­ ­ 4

optical path of the microscope, resulting in the desired circular circular desired the in resulting microscope, the of path optical tional polarization distortion is then corrected upon traversing the introducing a fixed ellipticity in the opposite direction. This inten will placed before the focus the at polarization become elliptical. To resulting compensate for this scrambling, the a optics, other and d in strain and birefringence system,and scanning the in laser to circular polarization. However, owing to non λ optics of polarization after the laser in polarization at the microscope focus, where these are the second set images are thresholded ( appear with greater intensity. This is shown more clearly when the fibers that are parallel to the direction of polarization (vertical here) multiple ­multiple channels (e.g., four or more). Although the Fluoview has with (excitation instruments commercial complex more than emission) and throughput optical higher a has experience, our in and, path optical simple a has microscope scanning/acquisition This stand. microscope upright BX61 system Olympus an scanning on mounted laser 300 Fluoview Olympus the use we able, system. Microscope/scanning (see PROCEDURE Step 21). stain’ ‘ring a achieving and symmetry cylindrical with specimen a imaging by found is condition This focus. the at polarization tions for circular polarization ( Figure in shown are images Representative gels. collagen fibrillar I type of using linear versus circular excitation, we imaged self preferred as it will excite all orientations equally. To show the effect ­polarization produced because of the electric dipole interaction rules. For is non signal little where direction, propagation axial the along than excitation power, and the intensities are directlyand intensity. comparable. We note that these imagesScale were acquiredbar, with the same 20 thresholded images ( using circular ( linear and circular excitation polarization. ( Figure 3 /4 (quarter We use a combination of two wave plates to achieve circular circular achieve to plates wave two of combination a use We c a 3

| , wherein uniform excitation is achieved in all fiber direc Comparison of SHG images of self-assembled collagen gels using - - a wave) plate first converts the linear polarization of the resolved SHG imaging, circularly polarized light is is light polarized circularly imaging, SHG resolved ) and linear polarization ( c λ and /4 plate and functions as a variable retarder byas a variable /4 plate functions and d ) show a different distribution of fiber alignment natureprotocols Fig. 3 Fig. 3 , bottom). Although many options are avail are options many Although d b a a b ). Linear polarization ( – ), and the resulting respective d Figure Figure ) Single optical sections acquired

| VOL.7 NO.4VOL.7 2 . Here a zero protocol - 45° reflections - | λ 2012 assembled /2 /2 plate is i Fig. 3 chroics - order | µ

659 m. b ) - ­ ­ ­

© 2012 Nature America, Inc. All rights reserved. used, but we have found the dichroic approach to provide better provide to approachdichroic the found have we but used, short and silver) or (dielectric reflector hard a alternative, an As below). described analysis polarization the for (although the design in this photograph has additional components The photograph in possible). as close as (located assembly photomultiplier the onto dichroic mirror (with bandwidth of ~100 nm) that directs the SHG initial separation from the laser, we use a 45° long wave pass (LWP) intensity.in highermagnitude ordersseveralof Forbe will which co the from signal the of separationefficient is direction forward the in detection SHG of challenge intensity.A SHG maximum for optimized condenseris the of height the and abilities, but this come will at the cost working of distance. water we use a 0.9 the excitation objective, where, for example, for 0.8 NA than higher somewhat with condenser a use to advantageous NA. becomes at a larger higher these between angle the Thus, it is because of the coherence, SHG does not have a corresponding point be approximated by the Abbe limit over are somewhat higher (~30%) than the theoretical limit, which can 700 nm and 2.5 0.8 the of nm 800 at functions spread point fluorescent beads and TPEF, subresolution we using have by measured example, the lateralFor and resolution. axial spatial good at for imaging tissues of several hundreds of micrometers in thickness the of near transmission for NA), optimized ×40,are0.8 for and mm 3 (e.g., distances ing work long with (~0.5–0.9) NA reasonable with ×60, to ×20 of of the microscope companies now offer dipping lenses over a range the use of long ient for detection of the forward SHG signal. Moreover, this allows fixed nal, user Moreover, imaging. SHG exter of use the allow that inputs has it only two channels, this is all that is required for forward 660 design. The forward SHG is emitted in a dual our in account into pattern emission SHG We the take also must together. frames tile then and NA higher a at image to prefer we tion, especially in the axial direction. Thus,resolu for lower larger fields also of view, and intensity SHG reduced concomitant and power peak reduced the both of because is This NA. ~0.5 be to effect raysdistal in the cone, where, for example, at 1.4 NA this is a 30% the for angle large the of because scrambled becomes excitation (~100–200 higher many tances (1–3 mm) and are ideal for deep tissue imaging. In contrast, Such medium water NA 0.8 ×40, a use we excitation for Typically, combination. objective/condenser every for calibrated be to need efficiencies collection relative the measurements, tive quantita For channels. requires collection backward microscope and forward SHG both complete the subresolution extract information, thus feature and SHG of coherence the exploit excitation/collection/detection. forward-backward SHG wavelength as an approximation. excitation same the at fluorescence use we but function, spread protocol We stress that forward SHG detection is not Kohler illumination,

| - VOL.7 NO.4VOL.7 6 - stage upright platforms, as this configuration is more conven 4 immersion condensers may offer increased light increased offer may condensers immersion . Lower NAs can be used, but we find a practical lower limit - defined detectors. Our SHG microscopes are mounted on - µ NA air condenser. For highly scattering tissues, oil m). In addition, at NA at addition, In m). - - - NA lenses have insufficient working distances distances working insufficient have lenses NA NA water working | µ 2012 m m laterally and axially, respectively. These values Figure |

- natureprotocols distance water - - immersion lenses have long working dis IR IR laser excitation. These lenses are ideal 4 is annotated to show these components

- > immersion objectives. Most

1, the polarization of the the of polarization the 1, √ 2 2 (ref. - - immersion objective. immersion lobed pattern - NA lens to be about about be to lens NA - - 63). We stress that propagatinglaser, pass filter can be be can filter pass - NA excitation - - backward gathering gathering 6 5 , where To - or ­ ­ ­ ­ ­ ­

focal detection as the desired light does not return along the the along return hitting before a pinhole not through passed it is nor path, does excitation light desired the as con from detection differs focal setup This epigeometry. nondescanned a in is This microscope. implemented most commonly scanning laser have somewhat lower sensitivity. photon either in used be also parameters are internal to the module. Other high controlsand offset in software the Fluoview are not used, as these gain voltage, PMT the scheme, this in that,scope.We emphasize micro every for validated be to needs approach this of linearity inputs used normally for regular PMTs (internal or external). The level, intoFluoview the directly (logic 0–5 plugged be can that V) discrimination, and output transistor and amplification gain, fixed have devices These detection. SHG cies ~40% of for blue wavelengths (400–500 nm) and are ideal for efficien quantum have photocathodes GaAsP the where 7421), photon use microscopes are pulses preamplified, individual discriminated and and converted mode, into saturation a digital in signal. run Our are (PMTs) tubes photomultiplier counting, For imaging. SHG for limiting be to this found however, not havedetection; we digital of range tional analog detection. A possible drawback is the lower dynamic convenmore the than sensitivity higher provides this as scopes, have the best transmission (~60%). (OD) for rejection the of laser. We have found that Semrock filters (20 nm FWHM) then provides an additional 4 or 5 optical density band objective.A the bright that suchsystem rail sliding separationhigherthroughput. with dichroicThe mountedis ona light-tight box. Figure 4 Backward SHG detection requires fewer modifications to a to modifications fewer requires detection SHG Backward single Weuse

dichroic mirror | Annotated photograph of the upright microscope, detectors and Long-pass - photon counting detection in our SHG micro SHG our in detection counting photon - pass filter for the desired SHG wavelength SHG desired the for filter pass - counting PMT modules (Hamamatsu, (Hamamatsu, modules PMT counting - counting or analog mode but will will but mode analog or counting - field can be used for focusing for used be can field - transistor logic (TTL) pulses Scan head - gain gain PMTs can Backward PMT PMT detector Forward detector ­ ­ ­ ­ ­

© 2012 Nature America, Inc. All rights reserved. order order to minimize signal lenses are placed before the detectors in both collectionlight channels in in detectors and stand scope detection. Toforward stray light, minimize we the enclose micro and band fluorescence detection. Next, the arc lamp is replaced with the PMT dichroic for SHG is placed in the infinity space as for conventional each). (~20–30% losses large have can which mirrors, scanning galvo the especially surfaces, fewer off reflected is signal the Second, used. be to PMT area the of whole the allows detection confocal the nondescanned where miss pinhole, would photons scattered First, factors. two of because tissue imaging in detection confocal over sensitivity increased greatly of advantage key the has This to mirrors. scan the returning before isolated is signal SHG the Instead, needed. to intrinsic is nonlinear the sectioning optical the because is This PMT. the This is achieved by replacing the standard standard the replacing by achieved is This excitation. this to respect with the specimen rotate then and one polarization, select to linear space infinity the in cube (PBS) to beamsplitting polarization is a place method precise more and second The zoom). lower (i.e., angles scan larger at significant more also are polariza errors tion); circular obtaining to analogy (in used is precompensation unless errors a with the polarization is rotated in the beam path and fixed is specimen the first, the In lent. equiva physically principle, in are, which This can be implemented by two methods, recorded. is successive images these of sity then rotated through 180°, where the inten with the long axis of a collagen fiber(s), and aligned is polarization laser the surement, respectively angle, helical pitch angle and the dipole alignment linear polarization excitation, where these yield data on the protein of constant for anisotropy function signal the analyzing a or polarization as laser intensity the measuring of form the in be can This intensity. image by or lengths fiber of visualization simple beyond information assembly structural extract to used be can microscopy. polarization SHG the software control should not be used. in offset and gain the and changed never is voltage the counting, single of case the For calibration. for used was as ment experi actual the for used be must offset and gain voltage, same pathways (e.g., different dichroics or is used,filters). detection analog the If two the of throughput spectral in differences any for fluorophore that emits near the SHG wavelengths, as this accounts to use two readily achieved fluorescentusing or a beads dye slide. We choose is and emitters isotropic with done be must calibrated. toThis be need detectors, the pathways, including detection two the of cies components. these of on placement the for details the detector input window. See the photographs in For quantitative F/B ratio measurements, the relative efficien relative the measurements, ratio F/B quantitative For To scheme, LWPthis the detection first appropriate implement λ /2 plate. However, this can lead to However,plate. lead /2 can this - - pass pass filter, where these are identical to those used for the photon excitation beads of (e.g., 15

excitation, and thus the confocal geometry is not not is geometry confocal the thus and excitation, 5,39,6 6 I te is mea­ first the In . - beam beam spot size so that it does not exceed Polarization - tight boxes. Weakly focusing focusing Weaklyboxes. tight ­ ­ ­

Figure 5 µ Condenser m) m) labeled with a - resolved SHG SHG resolved Figures

| Annotated photograph of the optical setup for the forward polarization-resolved detection. - Long-pass dichroic photon photon 4

and mirro 5 ­ ­ ­ r

tion and reduction of the spot size to passparallel theand 10 rapidly diverges. Thus, a telescopescheme isis not requireda Kohler forgeometry, collima the light from the condenser polarizationis not between the isolating dichroic mirror and the PMT than in the needednon polarization–resolvedis wardspace detection.more Here will be significantly less than that attained by Glan polarizers. film polarizers can be used, although the attained polarization purity sotropy, tion microscopy. stage a with circularly centered stage, as is often used for polariza of SHG, medium NA (approximately 0.6–0.8) and magnification magnification and NA (approximatelySHG,0.6–0.8) medium of considerations, as well as the double imaging of tissues are given in acquisition. Data analogous to that of the forward detection. manner a inimages thecollect thendetector; webeforethe these ropy,we use two GLPs oriented inorthogonal directions and place rotationstage under computer control. For backward SHGanisot the orthogonal polarization states or by mounting it in a the rail)motor relative drive to the laser can be rotated either manually between tooptimize thedetection. theGLPThe(alsoangle of mounted on railsystem. ThePMT assembly ispositioned ontranslationa stage of 200 0.8 the GLP and be incident on the 6 is chosen as it corresponds to the largest SHG response nents are measured relative to this excitation polarization.then This inangle successive images the SHG parallel relativeand perpendicular to the predominantcompo fiber axis (or respectively.on a Herefiber linearthepolarizationlaserfixed the is 45° at of by fiber basis), and GLP oriented parallel and perpendicularly to the laser polarization, where Figure The second polarization measurement determines the SHG ani - and 0.9and I - par and 50 β and 5 , which is given as follows: shows a photograph of the optical setup for the for the for setup optical the of photograph a shows Lens - - resolved setup described above. As the forward detection NAobjective condenser,and combinationtheusewe I - perp mm focal length singlet lenses mounted on a sliding correspond to the SHG intensity detected after a Typical data acquisition parameters for SHG SHG for parameters acquisition data Typical natureprotocols b = I I Table I I par - par rotational stage mm PMT photocathode. By using a Polarizer on motorized band-pass filter + − 3 - 2 Narro . Because of working lobe lobe spatial emission pattern perp perp w a

|

VOL.7 NO.4VOL.7 - mm clear aperture of Lens Forward PMTdetector protocol 3 | 9 2012 . Note that

distance |

(4) 661 - ­ ­ ­ ­ ­ ­

© 2012 Nature America, Inc. All rights reserved. (×20 to ×40) are most commonly used for imaging tissues. To tissues. imaging for used commonly most are ×40) to (×20 T 662 • • • • • • • • EQUIPMENT • • • for calibration Materials necessary • • Fixed to produce samplesknown SHG REAGENTS M averages quality. image to achievegood For polarization signals. 2–5 We noisy use and/or weak for typically needed is this acquisition time is required, where this increases with averaging if ~200 ~1 of sizes step minimum lens, this with sampling axial proper for Correspondingly, field. 200 × 200 about 0.8 for which necessary, is 2 zoom of minimum a criterion, Nyquist the satisfy a Number of images Pixel dwell time Field size Z Zoom NA Magnification P protocol dichroic mirror chosen used, canbe to match if theexcitation range Excitation dichroic mirror (Chroma Technologies, DM800/500). A different Electro priate eye protection near working when with Coherent MIRA 900 ! Ti:sapphire. compatibleA different with if pumplaserused canbe Coherent Verdi 10 Watt Nd:YVO least 100–200 used,can be but theNA atleast0.5andtheworking shouldbe distance at UmPlanFLOlympus ×20, 0.5NA; Olympus) Water tissue imaging. used,stand canbe afixed although BX61fixedOlympus FluoViewOlympus 300laser scanningmicroscope, or similar least 48inches ×72inches. inches. butA different used isolationtable at canbe shouldbe vibration dimensions 48inches ×96 isolationopticaltable of vibration Newport Note orused thatsmaller largercanbe beads respectively (FluoSpheres, Invitrogen), for forward Fluorescent (15 beads Di (theseare calibration preparedpolarization by astandard procedure Lipid for makingvesicles ( Starch homemade granules: Pollen slides for fluorescence grain (Carolina andSHGalignment Scientific) ATER

-step -step size arameter b CAUT -

le le 8 | - VOL.7 NO.4VOL.7 ANEPPS voltage µ - 3 3 immersion objectives LUMPlanFL (Olympus ×40, 0.8NA and I - m m in thickness, at 4 I ON optic Technology opticalisolator, or similar ALS |

Typical acquisition parameters. Use appropriate eye lasers. protection visible working when with µ - m for tissueimaging. NA ×40 magnification results in a field of view of of view of field a in results magnification ×40 NA | 2012 µ - - - m, with pixel size of ~0.4 ~0.4 of size pixel with m, F Ti:sapphire laser, or similar stage upright microscope.stage upright A different microscope sensitive dye (Invitrogen, cat. no. 3167) for staining lipids µ m) absorbing andemittingat430465nm,m) absorbing |

l natureprotocols - a - µ phosphatidylcholine, Avanti Polar Lipids)for µ s s per pixel, a minimum of ~15 min of 4 m are required. Thus, for tissues of of tissues for Thus, required. are m laser to pumptheTi:sapphire laser. - stage upright platform ispreferred platform stage for upright  - infrared lasers. R 512 512 × 512 pixels

CR ange of values - ×20 ×20 or ×40 I ! backward calibration. T

4–20 4–20 0.5–0.8 1–2 1–2 CAUT 10–200 µ I CAL m for a 512 × 512 512 × 512 a for m 2–6 µ Otherobjectives I µ ON m s Use appro - resolved

6 7

)

­ backward analysis. These can run in ImageJ or Fiji or be automated thresholding for measuring fiber lengths and division for forward intensities, measuring as such functions math image use data,we SHG the of analysis For packages. software external use mainly have some rudimentary data analysis capabilities, for flexibility we analysis. Data now been used for imaging of human skin intensity.signalhavemodalitiesnonlinear opticalother and SHG backward lower the of because smaller somewhat be will depths whole in ments note that backward detection can be used for morphological assess their axial extent in both the forward and backward directions. We tering tissues of ~200–400 most tissues, with Ti:sapphire excitation (700–1,000with a microtome),nm), or highlyfixed or scatlive tissues cut with a vibratome. For Specimens. slice anywithout axial drift. ments, as a series of images must be acquired from the same optical the setup is absolutely required for accurate polarization measure laser polarization, through 180° of rotation. Mechanical stability of of 10° every least at acquired typically are images measurements, that these processes are not specific to SHG but are integral to fiber measuring integral lengths and their distributions. are but SHG to specific not are processes these that commercial Improvision including Imaris. and packages We note with performed be can LabVIEW.MATLABrenderings or in 3D • • • • • • • • • • • • • • • IR viewer (ElectrophysicsIR viewer Electroviewer, cat. no. 7215, or similar) Laser power meter (Molectron Powermax, 500AD, or similar) renderingImaris software, or similar(Bitplane) MATLAB or LabVIEW software (or similar)for automated analysis ImageJ or FIJIsoftware (open access, USNational Health) Institutes of haverange. quantum thebest efficiency inthisspectral design, inExperimental asdescribed used butcan be GaAsP photocathodes Photomultiplier module(Hamamatsu, 7421) controlledcan be design) inExperimental manually asdescribed Pockels (Con Cell used,can be design. inExperimental as described GLP for laser power control (CVIPTY used, design. inExperimental asdescribed channels, respectively (CVIPTY Three GLPs for SHGanisotropy, one andtwo for theforward andbackward band Zero to match the laser excitation range. for power and polarization control. A different wave plate can be used, chosen Two zero same wavelengths. calibration.polarization the ittransmits A different if used canbe PBScube measurements forPBS cube polarization (CVIPBSH have the appropriate bandwidth, i.e., 10–20pass filternm for chosen100 to match the laser excitationBand range can be used but it needs to so asto match theexcitation range. (CVI SWPT Dichroic mirror theTi:sapphire for separating from residual pumplaser mirror chosen used, canbe soasto match if theexcitation range. dichroicDetection mirror (CVILWP T - - - order pass filter can be used, chosen to match the laser excitation range. pass filters (Semrock Brightline, 445/20 nm) - order λ /4 wave plate centered at 870 nm (CVI QWPO Specimens can be in the form of fixed slides (e.g., sliced - 00 λ Although commercial laser scanning microscopes microscopes scanning laser commercial Although - R /2 wave plates, centered at 870 nm (CVI QWPO - - animal imaging, in which the attainable imagingattainable the which in imaging,animal - optics, 350; adifferent Pockels or cell used power canbe 900). A different dichroic mirror chosen used, canbe if µ m in thickness can be imaged throughout - 10.0 - 425 - - 10.0 900 - 675). A different canbe polarizer - - 670 R  - 400). A different dichroic 

- CR in vivo 1064). A different polarizer

CR I - T fs laser pulses. I - I T 670 CAL I - CAL 870 23,2 - A different PMT 980 4 A different band - . 10 - - 100) and 4). A different - 870 - 10 - 2) - - ­ ­ ­

© 2012 Nature America, Inc. All rights reserved.  3| or anIRsensing card orIRviewer. 2| with alllasers. ! alignment of the laseratsafepower. Ti:sapphire laserfor initial alignment purposes. Thisaffords 1| General preliminary alignment scope. Day-to-day operations are described in Steps 9–36.  PROCE ?  12| 11| 10| turret. the objective in slot empty an through centered is it that so microscope, the through laser the realigning by begin slightly, beam the walk can optics control power the As conditions. polarization different under measurements for subsequent first performed be must that components specific of deletion or insertion either for are rectangles rounded the in 9| P 8| 7| transmission byrotating the angle of the rotation stage. Next, insert the and aGLP(670–1,064nm).Forthe latteroption, firstinsert the GLPand the laser power meter and maximize the laser 6| location is not crucial, but the back aperture of the objective needs to be filled for best resolution. depend on the laser divergence and the distance. The precise for the lens between the laser and the scanner will the scan head is ~1 mm in diameter. The proper location in the beam path such that the resulting spot entering 5| this alignment to essentially park the beam. this procedure. In this case, use high digital zoom (~10) for may need to be on with the shutter open and scanning for an empty slot. Note that on some microscopes the instrument to center the beam at the exit of the objective turret using 4| ing pairsof apertures inthe beampath( scan head parallel inbothplanes. Thisisfacilitated byplac

reliminary reliminary image acquisition

CAUT TROU

CR CR CR Adjust the condenser height sothatitisnear the levelof the bottomside of the specimen. Turn off the bright-field source. Placethe specimen onthe stage and find the focus withthe desired objectiveinbright–field mode. Insert the NDfilter( See See Adjust the rotation of the Remove the NDfilter. For powercontrol either follow the manufacturer’s specificinstructions for alignment of a Pockels cellorusea Insert the long distance focal length lens (1–2 m) Use the last turning mirror before the scan head entrance Align the laserinto the entrance of the scanhead. Align the laserthrough the optical isolatorwithpaper I I I T T T D I I I I B ON URE CAL CAL CAL LES Figure Figure Use appropriate eyeprotection when working Steps 1–8 describe the initial setup of the micro

H STEP STEP OOT 7 Thisisnot Kohler illumination, and the height of the condenser iscrucial inthe following alignment. It isimportant thatthe laserenter the for a flowchart of the day-to-day alignment and operation outlined in Steps 9–36. The directions directions The 9–36. Steps in outlined operation and alignment day-to-day the of flowchart a for I N G

>2 OD)atthe outputof the λ ● /2 platetoachieve transmission of 1–10mWthrough the GLP. ●

T T IMI IMI N N Fig. G G 2–4 d 2–4 d 6 ).

- - rail with apertures used for alignment. before the scan head, showing the polarization control optics and the optical Figure 6

| Annotated photograph of the optical layout of the laser pathway λ /2 platebefore the GLP. natureprotocols diaphragms Iris

| VOL.7 NO.4VOL.7 λ /4 waveplate protocol λ /2 waveplate | λ 2012 Polarizer /2 plate

|

663 © 2012 Nature America, Inc. All rights reserved. linear polarization; thisisagainverified using the PBScubebytaking outthe 20| transmitted. and reflected power 50% be will there where cube, PBS the with state polarization this verify meter, power 19| measured poweronthe powermeter. 18| reflects horizontal and vertical polarization, respectively. 17| the output of the objective (assuming 15| center of the PMTassembly. 14| the mirror surface. viewer) and the spotisnot larger than the dichroic (using anIRcard orIR height sothatthe laseriscentered on the condenser. Readjustthe condenser 13| Initial setup procedures are given in Steps 1–8. approximate times and corresponding step numbers.procedures for typical SHG imaging of tissues with Figure 7 664 C ? can be determined by looking 16|at the histogram and also using the Hi-Lo feature in the ? lookup tables, which shows saturatedmust be optimized for every pixelsobjective. in red. condenser and subsequent alignment condition. Note that the height of the mirror mount assembly to achieve this height of the condenser and the dichroic the alignment into the microscope, the micr by using the translational stage on the grain) is even across the field of view of a given object (e.g., a single pollen the PMT. Further verify that the intensity as seen in bright-field, is centered on Verify that the field of view at zoom 1, controls on the dichroic mount ( by adjusting the vertical and horizontal the height of the condenser, and then optimize the SHG intensity by adjusting pollen grains or starch granules. First these could be fixed objects such as produces SHG, where for simplicity, image with a known specimen that 0.8-NA excitation), acquire an SHG protocol ircular polarization alignment and calibration

TROU TROU

Adjust the power into the microscope such that the full dynamic range of the integrator is used. In the Olympus microscope, this With approximately 10–50 mW at Insert the Insert the Use aPBScubetoestablishthe laserpolarization before and afterthe isolator, where thisoptic transmits and Center the residual laseronthe Slide the dichroic mirror under Rotate the the Rotate o | VOL.7 NO.4VOL.7 scope. It may be necessary to iterate

| B B Flowchart of the day-to-day operational LES LES H H OOT OOT | λ λ 2012 λ /4 platesuch thatthere is no optical rotation; thisisverifiedusing the PBS cube byobserving no change in /2 plate(acting asavariable retarder) before the /4 plate to obtain circular polarization; this will be achieved by 45° rotation (nominally). By using the the using By (nominally). rotation 45° by achieved be will this polarization; circular obtain to plate /4 I I N N G G | natureprotocols Fig. 5 ).

GLP beforethePMT(5min). infinity space,removethe beamsplitting cubeinthe For laterSteps22A,25 ● Add thepolarization

T IMI and adjustlaserintensity,scanspeeddigitalzoom(10min).Steps30,3233 Turn onthePMTsandtestimageacquisitionparameters.Finddesiredfieldofview, Start Fluoviewon perpendicular emissionpolarization(anisotropymeasurement),orforforwardand Put thespecimenslideonmicroscopestageandbringittovisualfocususing N Determine theaveragingnumberandacquireSHGimagesforsamplestageat backward channels(forward-backwarddetection)(1–10min).Steps34–36 G various angles(excitationpolarizationmeasurement),orforparalleland 3–6 d Optimize thelaserandverifythatismode-locked(5min) Yes Turn onthelaserandallowtimetostabilize(20min) λ × /4 plateand rotate itsothatthere isno rotation of the microscope usinganIRviewer(5min).Step 10 zoomandcheckthealignmentoflaserbeamthrough bright-field mode(5min).Step10 ForlaterSteps18,20,25 polarization, addtheGLP Remove the before thePMT(5min). plates forlinearlaser polarization? Vary relative λ excitation /4 plate. Yes λ /4 and λ /2 anisotropy? Measure No 9 GLP beforethePMT(5min) Add the For laterSteps18–20,25 polarization, removethe for circularlaser λ /4 and No λ /2 plates

.

© 2012 Nature America, Inc. All rights reserved. laterally shifted relative tothe laser. and collimating lenses, the desired SHGmay beslightly that becauseof chromatic aberration inthe condenser or starch granules) tooptimizethe collection path. Note 27| the PMTassemblywithrespect tothe residual laser. 26| passes the clearaperture of the GLP. Adjust their positions until the beamiscollimated and 25| 24| 23| A sample such as mouse or rat tail tendon.When is the sample time, later a at zation circularity one can use a well-aligned the at focus. state routine a polarization For check of polari fairly precise controlstrates that this required is attain to rotationnot is achieved. circularpolarization demon This Theproperwhereas rotation and of20°, 10° at 30° at is tained(the ofperimeter the vesiclesdo even). not appear rotation),the at focusnot circularpolarization is main no with precompensation that the with images aroundthe perimeter. shown is in This rotatethe keeping While the Di-8-ANEPPS. sensitive differentmembrane stainingdyes,the used voltage- we but membrane a with stainingdye. with labeled be These can 21| (B) ( 5×) toavoid polarization distortions. plate external tothe microscope inthe laserexcitation path.Thiscanonlybeusedathigh zoom(e.g., greater thanabout can beusedatany digital zoom.Inoption B,the specimen isfixed and the polarization isselectedby rotation of the zation. Thisdoes not sufferfrom any birefringence inthe optical paththatcandistortthe polarization purity. Further, this In option A,the specimen ismounted onacircularly centered rotation stage and rotated withrespect toafixed laserpolari methods, wherein option Aismore precise thanoption B. 22| L across the image. observed be the rotatedin focal plane, no change intensity SHG in must A inear inear polarization selection nisotropy alignment (iii) Verify the polarization atthe focus byusing the PBScube. (iii) Mount the desired specimen onthe circularly centered rotation stage and rotate ittoacquire images. (iv) (ii) Withno rotation of the (ii) Insert aPBScubeinto the filtercubeinthe infinityspace. ) (i) Reversethe order of the (i) Center the residual laser on the two collimation lenses. Acquire animage of atestobject(e.g., pollengrains Byusing the translation stage, approximately center Adjust the position of the detection dichroic mirror asdescribed inStep13(see Choose the desired linear polarization atthe focus asdescribed inSteps17–22. Selection of linear polarization canbeachieved bytwo Acquire SHG or TPEF images Acquire TPEF or ofSHG giant vesicleslabeled S S election by external election by rotation of themicroscope stage at high zoom( Adjust the Set the

acquired over a range a acquiredover of angles of rotation. We note λ /2 plate until the plate stainingvesiclesshow /2 even λ /2 and λ /2 plateuntil the desired polarization isachieved and acquire images. Note thatthisapproach worksbest

>5), where the scanangle issmall, thereby resulting inlessellipticity. λ ● /4 platessothatthere isno rotation when using the PBScubeasdescribed inStep20.

T

IMI  /2 plate λ N λ ● /4 plate, choose the desired polarization withthe G /2 and

T 2–4 d IMI N Figure λ G λ /4 plates. 2–4 d /2 plate (0 degree (0 plate /2 λ /4 plate fixed, plate /4 8 for TPEF -

-

- plot and fit images are shown for 0°, 45°, 90° andFigure 135°. 9 Scale bar, 50 a plate. The correct position is at 20 degrees, at which awith ring the stainmembrane is achieved. staining dye Di-8-ANEPPS with the rotationFigure of 8 the 90 0 ° °

| SHG intensity dependence on the laser polarization. ( 3 wave plate orientation 9 from optical sections from 37 angles.

17.5 | 20 10 λ 0 Two-photon excited fluorescence images of giant vesicles labeled /2 ° ° ° ° 135 45 ° ° λ /2 plate. fluorescence Two-photon Fig. 5 image natureprotocols ) b . 10 20 30 40 4 0 wave plate orientation Polarization angle(degrees) 22.5 45 40 30 λ /2 9 5 ° ° ° °

| VOL.7 NO.4VOL.7 µ m. ( 0 a b ) Representative protocol ) The resulting fluorescence 135 Two-photon image | 2012 λ 180 λ /2 /2 |

665 - © 2012 Nature America, Inc. All rights reserved. laser polarization, either rotate the specimen on the centered 35| as thiswilleliminate high-frequency noise. 34| the integration electronics. the laserpower(Step6)tooptimizethe dynamic range of 33| criterion for the chosen NA. 32| 31| 30| Data acquisition counting. photon single- for used not are controls These step. calibration the in used those as measurement desired the for same the exactly be must offset) and gain (voltage, settings  0.9-NA condenser. intensities. In our microscope, this gives a forward/backward29| calibration factor of 1.025 for the ×40, 0.8-NA objective and and thiswillleadtoincorrect calibration factors. the forward and backward directions. Ensuring single particles isimportant, asaggregates may not beisotropically emitting 28| measurement and for everydifferent objectivelens. this alignment, and thisoptimization must bedone for every  17 was tissue into depth The polarization. laser the to perpendicular and parallel oriented GLP the with shown are tendon mouse for 10 Figure 666 overall F/B ratio. forward and backward SHG images, the calculated F/B ratio image and the that equals 2 and the small offset, eps (the floating-point relative accuracy for MATLAB field of view. Note the highlighted lines for the calibration factor, 1.025, calculate F/B ratio pixel by pixel and the overall F/B ratio for the entire collagen gel using MATLAB. ( Figure 12 C protocol alibration alibration of the forward and backward detection paths

µ CR CR

Integrate the resulting fluorescence intensities in ImageJ or FIJI, and then take the ratio of the forward and backward To measure the dependence of the SHGintensity onthe Use 2–5averaging ifthe SHGsignal isweakand noisy, Take one optical section with the desired zoom to adjust Setthe zoomand axial stepsizetosatisfythe Nyquist Placethe desired specimen onthe microscope stage. Referto Byusing sparsefluorescent beads(ensuring no m. Scale bar, 15 15 bar, Scale m. | VOL.7 NO.4VOL.7 I I T T I I

CAL

CAL | | Forward-backward analysis of SHG image from a fibrillar Representative SHG polarization anisotropy images images anisotropy polarization SHG Representative

−

52

), ), to avoid division by zero. (

STEP T STEP able | 2012 Parallel

µ The height of the condenser iscrucial for ● If analog detection is used, the PMT PMT the used, is detection analog If m. 3

T | for typical data acquisition parameters.

IMI natureprotocols a ) ) Screenshot of the MATLAB script used to N G 1 d b ) ) MATLAB output showing the Perpendicular

aggregates) emitting near the SHGwavelength, acquire TPEFimages in

●

T IMI image (1 unit measurement result showing the length in fiber the ofunit relative the top to measure its length using the (in the and red is a circle) selected is freehand drawnline (yellow) on SHG image; center, thresholded image to used identify One fibers. fiber Figure 11 b a N G 2–4 d

Raw data | Using ImageJ to lengths.measure fiber rawcollagen Left,

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© 2012 Nature America, Inc. All rights reserved. Steps 37–39,data analysis: 2–3d Steps 30–36,data acquisition: 1d(See Steps 28and 29,calibration of the forward and backward detection paths: 2–4d Steps 23–27,anisotropy alignment: 2–4d Step 22,linear polarization selection: 2–4d calibration: 3–6d Steps 17–21,circular polarization alignment and Steps 9–16,preliminary image acquisition: 2–4d Steps 1–8,general preliminary alignment: 2–4d ● Troubleshooting advice canbefound in ? optical section with the average maximum intensity. over the field of view or region of interest using ImageJ or FIJI. The data for each optical 39|series are self-normalized to the ( value withthe calibration factor from Steps28and 29.See The F/Bcanthen becalculatedfor the entire field onapixel-by-pixel basisand averaged overthe field of view. Correct this 38| ? the left and center images are the raw and thresholded images,shows respectively, ImageJ screenshots and the for output the ofprocedure fiber sizes for is agiven single on theSHG opticalright. section from a self-assembled fibrillar collagen gel, wherein the background noise. This must be calibrated for the field ofdetermine view, as ImageJ the has lengths. an internal pixel-size We choose calibration. the 37|threshold level on the basis of discrimination Data analysis of signal from fibrillar collagenous tissues against ? tendon are shown in take successive images withthe GLPoriented parallel and perpendicular tothe laserpolarization. Representative images for 36| mouse tendon for several polarizations are shown in stage or by using the external T a Fig. 12 37 36 16 15 12 S

TROU TROU TROU

tep b T le le Use ImageJ or FIJI to measure fiber lengths. Use the threshold function to identify fibers and then the freehand tool to For attenuation measurements (the relative SHG intensity as a function of depth into the tissue), integrate the intensity IMI Use MATLAB orImageJ tocalculatethe F/Bratio. Add asmall offset inimage math toavoid division byzero. To measure the SHGanisotropy, choose the desired laserpolarization (aligned withthe fibers of interest), and then 4 4 N B B B | a LES LES LES G

), the SHGimages ( Inconsistent measurements ­anisotropy specimen Low anisotropy from known high- Photodamage of stained specimens Uneven field in forward SHG No forward SHG signal P Troubleshooting table. roblem H H H OOT OOT OOT ●

T I I I IMI N N N G G G Figure 1 N G 2–3 d Fig. 12 0 λ . /2 plate (Step 22). Acquire images atleastevery10°of rotation. Representative images for b ), the F/Bimage and the resulting average overthe field of view. T Fig. able 7 for abreakdown of the times required for specific measurements) 4 . Background light is reaching the detector polarization is incorrect Angle of GLP relative to the excitation ­photon resonance of stain Too much power or too close to two- ­parallel Alignment into the scan head is not illumination Condenser is closed from bright-field P

ossible reason Figure 9 Figure 1 a , and the resulting plotand fit 2 for representative screenshots for the MATLAB script natureprotocols Take background images and subtract ­perpendicular to the laser polarization to verify where the GLP is parallel and Use a PBS cube after the condenser ­wavelength Lower the power or change the Realign using a known specimen Open the condenser S olution 3 9 are shown in

| VOL.7 NO.4VOL.7 Figure 9 protocol Figure 1

| 2012 b . 1

|

667 © 2012 Nature America, Inc. All rights reserved. In contrast, littlemodulation of the response, ashifted focus of the microscope isthe desired linear polarization. and demonstrates thatthe polarization of the laseratthe of collagen. Thisprofile ischaracteristic of typeIcollagen, we developed analysis are shown in and canbeused. although other rendering software programs are available tion of depth). The renderings were performed withImaris, well asthe relative attenuation (i.e., the intensity asafunc attributes canthen beused todetermine the F/Bratio as tive forward and backward results. The depth-dependent collagen gels are provided in Representative 3Drenderings of SHGimages from fibrillar ANT 668 3. 2. 1. com/reprints/index.html. http://www.nature. at online available is information permissions and Reprints Published online at http://www.natureprotocols.com/. interests. CO P.J.C., S.P., O.N. and others primarily developed the SHG tissue-imaging protocols. developed the analysis scripts; S.P. and O.N. constructed the first instrument; constructed the current instrument, acquired the data for the manuscript and AUT Institutes of Health grant no. CA136590. A fibrillar collagen gel. Inthe latter, the fieldis noteven, and the dichroic mirror ( larization analysis path.The leftand right panels show properly and misaligned images, respectively, from aself-assembled laser polarization orincorrect polarizeralignment. Finally, SHG polarization anisotropy. Ifloweranisotropies are obtained thanthose shown here, itwouldbeindicative of incorrect we showed thatnear-unity valuesfor tendon were obtained tropy is rapidly reduced by scattering corresponding to complete alignment. However, the aniso β for averythinfiber(lessthanone scattering length), the length inthe visible/near-UV spectral region depth of 17 or onapixel-by-pixel basis. These images were acquired ata obtained byeither integrating overthe whole field of view We note thatfor thisregular structure, similarresults are parameter tendon are givenin polarization scrambling orrotation atthe focal plane. response orasymmetric response wouldbeindicative of protocol c valuefor tendon wouldapproach the limiting value of 1.0,

k M Analogously, representative anisotropy images from Representative SHGpolarization-dependent images and H no

PET principles and applications to disease diagnosis. disease to applications and principles Biotechnol. organisms. and tissues cells, in arrays biomolecular visualizing (2011). 13–26 applications to diseases diagnostics. diseases to applications Campagnola, P. Second harmonic generation imaging microscopy: imaginggeneration harmonic P.Second Campagnola, for microscopy imagingSecond-harmonic L.M. Loew, P.J.& Campagnola, microscopy:generation harmonic SecondC.Y. Dong, P.J.& Campagnola, | I OR VOL.7 NO.4VOL.7 C w I I

N CONTR le PATE G G FI dgm β NANC

IB D withequation (4)yielded avalueof ~ ents µ 21 3 UT m, which corresponds toaboutone scattering RESULTS 9 , 1356–1360 (2003). 1356–1360 , | and are related tothe I 2012 I We gratefully acknowledge support under US National AL ONS I

NTERESTS P.J.C. primarily prepared the manuscript. X.C. Figure 1 |

natureprotocols Figure

The authors declare no competing financial declare no The financial authors competing 0 9 Figure 1 . Calculating the anisotropy . The data are fittothe model Anal. Chem. Anal. 58,6 3 α 8 , showing the respec -helical pitchangle . Indeed, through optical clearing, which greatly decreased the scattering coefficient,

Laser Photonics Rev. Photonics Laser 83 , 3224–3231 (2011). 3224–3231 ,

2 9 . We note that

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. Tendon isthus anexcellent choice of tissuefor calibration of 11. attenuation data. Scale bars, 40 These images are used for calculating forward/backward ratios and ( Figure 13 10. 9. 8. 7. 6. 5. 4. Scale bar, 40 b the analyzing Glan-laser polarizer, whereas part of the field is missing in ( Figure 14 a a because the SHG signal does not pass the clear aperture of the polarizer. a a

, , b b

4 ) The forward and backward results are shown in ) The image in panel Biophys. J. Biophys. simulation. and experiment imperfecta: osteogenesis state diseased the of microscopy.imagingGeneration Harmonic Second by studied cancer ovarian in matrix extracellular the of Prev.Biomarkers Epidemiol. Cancer microscopy.multiphoton with evaluated cancer ovarian of biomarkers (2005). tumors (2011). carcinoma. breast human in survival invasion. local facilitates interface J. Biophys. tissues. biological in proteins structuralendogenous of imaginggeneration USA Sci. Acad. Natl. Proc. generation. harmonic second and fluorescence nativemultiphoton-excited Lacomb, R., Nadiarnykh, O. & Campagnola, P.J. Quantitative SHG imaging SHG P.J.Quantitative Campagnola, & O.Nadiarnykh, R., Lacomb, Nadiarnykh, O., Lacomb, R.B., Brewer, M.A. & Campagnola, P.J.Alterations Campagnola, & Brewer,M.A. R.B., Lacomb,O., Nadiarnykh, opticalEndogenous Utzinger, U. & Brewer,M.A. N.D., Kirkpatrick, E. Sahai, M.W.Conklin, P.P.Provenzano, P.J.Campagnola, W.R.Zipfel, shows the importance of the alignment through the po

| | 3D renderings of SHG images of a fibrillar collagen gel. Forward SHG anisotropy images from a fibrillar collagen gel. in vivo in µ et al. et m.

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