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Two-Photon Microscopy and Imaging

Two-Photon Microscopy and Imaging

61

Two-photon and Imaging

Patrick Theer, Bernd Kuhn, Dorine Keusters, and Winfried Denk Max Planck Institute for Medical Research, Heidelberg, Germany

1Introduction63 1.1 64 1.2 Increased Penetration Depth 66 1.3 Reduced Photodamage and Photobleaching 67

2 The Multiphoton 67 2.1 Sources 68 2.2 Excitation Pathway 69 2.3 Final Focusing and Resolution 69 2.4 Detection 70

3 Limitations 71 3.1 Temporal Resolution 71 3.2 Spatial Resolution 72 3.3 Penetration Depth 73 3.4 Photodamage and Photobleaching 74

4 Fluorophores 74 4.1 Comparison of 1PA and 2PA 74 4.2 Intrinsic Chromophores 75 4.3 Synthetic Dyes 76 4.3.1 Ion Indicators 76 4.3.2 Voltage-sensitive Dyes 76 4.4 Genetically Encoded Fluorophores and Indicators 77

5 Applications 77 5.1 Neurobiology 77 5.2 Calcium Imaging 79 5.3 Imaging of Metabolic Activity 80

Encyclopedia of Molecular and Molecular Medicine, 2nd Edition. Volume 15 Edited by Robert A. Meyers. Copyright  2005 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim ISBN: 3-527-30652-8 62 Two-photon Microscopy and Imaging

5.4 Photoactivation (Uncaging) 80 5.5 Human Diseases and Therapy 81

6 Future Directions 82

Bibliography 83 Books and Reviews 83 Primary Literature 83

Keywords

Confocal Microscopy (CM) Microscopy technique where optical sectioning is achieved by using a detector pinhole that is confocal to the excitation to reject the out-of-focus signal.

Fluorescence Light-emitting process of special , called fluorophores, after excitation with photons of higher energy.

Laser-scanning Microscopy Microscopy technique, in which the light source is focused to a -limited spot and the image is formed by scanning this spot across the sample while sequentially recording the signal generated at each position.

Multiphoton Absorption (MPA) Process where two or more photons are absorbed simultaneously to excite a transition with an energy equal to the sum of the energy of the photons.

Multiphoton Microscopy (MPM) Microscopy technique where the contrast mechanism is based on a multiphoton absorption process. Usually, but not always, the detected signal is fluorescence.

Two-photon Microscopy (2PM) Most widely used variant of multiphoton microscopy.

Whole-field Detection Detection scheme for -scanning microscopy where the light from the whole field of view is detected.

Wide-field Imaging Imaging technique where the whole field of view is illuminated simultaneously and imaged onto a spatially resolving detector, for example, a charge-coupled device (CCD) camera. Two-photon Microscopy and Imaging 63

 Multiphoton microscopy (MPM) is an imaging technique that employs signals, such as fluorescence, generated by a multiphoton absorption (MPA) process as its contrast mechanism. Compared to other fluorescence microscopy techniques, MPM often has a higher sensitivity, superior penetration into scattering tissue, and causes less photodamage to the sample and less photobleaching of the fluorophore. These advantages make MPM the method of choice for optophysiological experiments and also make MPM the only technique available for high-resolution in vivo imaging deep within scattering tissue, such as the brain. The two-photon microscope (2PM) is the kind of MPM used in the overwhelming majority of applications since it has virtually all of the specific advantages of MPM. Recent advances in staining techniques including the booming development of genetically encoded fluorophores keep increasing the field of actual and possible MPM applications.

1 a signal – such as fluorescence – that is Introduction generated by absorption of light from the beam. Over the past 15 years, multiphoton mi- Molecules that can easily absorb light croscopy (MPM) has developed into an generally have a structure where sev- indispensable imaging tool in the life eral of the are connected sciences. MPM has higher sensitivity, su- by conjugated single–double bonds. As perior penetration into scattering tissue, a consequence, the most weakly bound and often causes less photodamage in sam- are delocalized and thus inter- ples than alternative imaging techniques act easily with the electromagnetic field. such as (CM). This Near-ultraviolet or visible light is usually means that MPM – combined, in partic- sufficient to promote the outermost elec- ular, with one of many new genetically tron to an excited level (Fig. 1). In general, encoded and functional dyes – has become the molecule is excited electronically and the method of choice for in vivo imaging vibrationally, with the latter excitation de- − in strongly scattering tissues such as skin, caying very fast (∼10 12 s). From the state muscle, and tumors, and even deep within reached after vibrational relaxation, which intact organs such as kidney, pancreas, is still electronically excited, the molecule lymph nodes, and brain. then relaxes within nanoseconds (10−9 s) This article provides an overview of func- to a state that is only vibrationally excited. tional principles, instrumentation, and The energy difference can be carried away some of the applications of MPM. Fur- by fluorescence emission of a photon. ther details can be found in the books and The difference between one photon reviews and primary literature listed at the (1P)-excited and multiphoton (MP)-excited end of this article. fluorescence microscopy is that molecular In laser-scanning microscopy, a three- absorption is of a single photon in the first dimensional image is generated by scan- case and a photon multiplet in the second ning the position of a tight laser focus (Fig. 1). In a 1P process, the photon energy around in the sample while recording, equals the energy difference between the as a function of the focus position, ground state and an excited state. Light 64 Two-photon Microscopy and Imaging

Fig. 1 Principle of one-photon and Excited two-photon excited fluorescence. In 1PE state fluorescence, a molecule is brought to the excited state by absorbing a single One-photon Two-photon (blue) photon. Vibrational relaxation excitation excitation bringsthemoleculetothelowest excited state, from which it returns to the ground state by emitting a Virtual state longer- () photon. In 2PE fluorescence, the molecule is brought to the excited state by simultaneously absorbing two NIR (red) photons. The molecule again vibrationally relaxes, and emits a fluorescence photon from the same Ground level as in 1PE fluorescence (see color state plate p. xxv).

with a quantum energy less than the gap 1.1 between the ground state and the lowest Optical Sectioning lying electronically excited state cannot ex- cite the molecule by a 1P process, but if the In 1P microscopy, the signal generated is proportional to the light intensity (the light intensity is sufficiently high, multi- power divided by the beam area: I = P/A). ple photons of lower energy can cooperate As a result, the signal generated in each and combine their energies to promote the slice of a sample depends on the slice molecule into the excited state. For exam- thickness, but not the distance from the ple, two 800 nm photons can excite a tran- focus, since variations in the beam area sition that normally requires 400 nm light. are, as long as the power stays constant, Onemaypicturethisprocessasthefirst exactly balanced by inverse variations in photon exciting the molecule to a virtual the excitation rate per molecule. When state from where the second photon then imaging thick samples, the light from brings the molecule into its (real) excited the focus is thus always contaminated, in state. For this process to occur, the two fact, dominated by fluorescence generated photons have to arrive at the fluorophore above and below the focus. This is the within the lifetime of the virtual state, bane of wide-field fluorescence imaging = −15 which is less than 1 fs (1 fs 10 s) of 3-dimensional (3D) samples. In CM, as defined by the Heisenberg uncertainty this problem is solved by placing a ≤ principle (τ ¯h/E). The likelihood that pinhole in front of the detector. The two photons arrive at a fluorophore almost pinhole is confocal with the excitation simultaneously is proportional to the in- focus and rejects light that did not tensity squared and is all but negligible for originate from that focus (Fig. 2). This light sources other than a focused laser. property of the confocal microscope, called Therefore, two-photon absorption (2PA) optical sectioning, vastly improves the was observed only after the invention of the ability to obtain 3-D fluorescence images, laser. It is the strongly superlinear depen- particularly of thick biological samples. dence on the light intensity that gives 2PM The first nonlinear optical microscope (and MPM in general) its main advantages. used the second-harmonic signal and Two-photon Microscopy and Imaging 65

Focal plane

(a) (b)

PMT PMT Pinhole

Lens

Dichroic Dichroic mirror Objective Sample Sample

Focal plane

(c) (d) Fig. 2 Optical sectioning in one-photon confocal and in two-photon microscopy. (a) With 1PE, fluorescence is generated throughout the illumination cone. For 2PE (b), because of the nonlinear intensity dependence, fluorescence generation is limited to a small volume around the focus. In CM, optical sectioning is achieved by placing a confocal pinhole in front of the detector. This pinhole rejects (out-of-focus) fluorescence that is generated above and below the focus (c), fluorescence generated by scattered excitation light (e), and in-focus fluorescence that is scattered on its way to the detector (e). Because no out-of-focus fluorescence is generated in a two-photon microscope, no pinhole is necessary (d) and even fluorescence that is scattered on its way to the surface can be detected (f ). 66 Two-photon Microscopy and Imaging

PMT PMT Pinhole

Lens Lens

Dichroic Dichroic mirror mirror Objective Objective Sample Sample

Focal plane

(e) (f) Fig. 2 (Continued)

wide-field imaging. Scanning the focus, Common to scanned-focus nonlin- which was introduced later, strongly en- ear is that they provide hances the focal but not the out-of-focus excitation-based optical sectioning. This signal, and thus provides optical sec- is because, different from the linear (1P) tioning. The hopes placed on harmonic case, the total excitation in a slice in- imaging for biological specimens were, tersecting the beam strongly drops with however, not fulfilled, at least initially. distance to the focus (Fig. 2). In fluores- Two photon–excited fluorescence mi- cence microscopy, excitation-based rather croscopy proved to be much more imme- than confocal optical sectioning is par- diately applicable to biological problems, ticularly helpful since photodamage and due, first, to the central importance of flu- photobleaching are enormously reduced orescent labeling in biological microscopy (see below). (see Sect. 4) and second, to the differences in the physical characters of fluorescence 1.2 and harmonic generation. The main dif- Increased Penetration Depth ference being that harmonic generation is a coherent process, where field strengths MPM can generate high-resolution im- rather than intensities add, and the gener- ages from focal planes deep inside the ated signal depends on the square of the tissue, much deeper than CM, because chromophore concentration, and is liable signal is generated only at the focus allow- to interferences effects; in fluorescence, ing scattered fluorescence to be collected which is incoherent, light from different without loss of resolution or optical sec- fluorophores does not interfere with each tioning. In CM, only light passing through other and the signal increases linearly with the confocal pinhole is detected. However, the fluorophore concentration. due to scattering, the fraction of the signal Two-photon Microscopy and Imaging 67 originating from the focus that does pass light-induced damage to the sample the pinhole in the CM decreases exponen- (photodamage) or the fluorophore tially with imaging depth. Compensating (photobleaching), which in 1PM occur for the loss in signal by increasing the ex- throughout the sample even though citation power in CM in most cases would information is only gained from the focal lead to an unacceptable increase in photo- slice. In MPM, damage is confined to damage (see below). A further reason for those fluorophores (and their immediate the deeper penetration of MPM is that, vicinity) that do provide information. for the same fluorescent label it uses ex- A reduced relative MP absorbance citation light with a wavelength that is of intrinsic fluorophores may further longer (up to two times for 2PM) and contribute to the vastly improved tissue hence scattered much less. Occasionally viability sometimes seen even in tissue helpful is the larger separation between that scatters only little. the excitation and emission in MP excited fluorescence, which allows a 2 more spectrally complete detection of the The Multiphoton Microscope fluorescence.

1.3 The layout (Fig. 3) of a generic MPM is Reduced Photodamage and very similar to that of a laser-scanning Photobleaching CM, from which an MPM can often be constructed by modification. In this Protracted and high-resolution CM section, the components of an MPM will imaging is often limited by excitation be discussed individually.

NIR Detector fs-laser Barrier filter

Collection lens Intensity control

x-y scan Dichroic mirror Scan Tube lens lens Objective

Sample on x-y-z stage

Fig. 3 Setup of a generic two-photon microscope. Light from an ultrafast laser or laser amplifier is focused to a diffraction-limited spot by an objective. The beam is raster-scanned across the sample and the fluorescence generated at each position is collected by the objective, separated from the excitation light by a dichroic mirror and additional filters either in a transmission or reflectance configuration (see inset), and detected by a whole-field detector. 68 Two-photon Microscopy and Imaging

2.1 where indicates an average over time, δ Light Sources is the two-photon absorption cross section (often expressed in units of Goppert-Mayer¨ − The development of reliable ultrafast laser (GM); 1 GM = 10 50 cm4s/photon), and sources operating in the near infrared C is the fluorophore concentration. For a (NIR) coincided with the invention of spot with area A, power and intensity are MPM and has been an important factor related by: in the broad adoption of MPM, since with P(r) continuous wave (cw) , even 2PM I(r, t) = .(2) is only marginally possible for brightly A labeled samples. Today the vast majority of For a square pulse with pulse length τ, MPMs use mode-locked titanium sapphire the ‘‘two-photon advantage’’ is lasers (Ti:sapphire, Ti:Al2O3), which can  be tuned from about 680 to 1100 nm, 1 T I(r, t)2dt allowing 2PE of almost all relevant labels, I(r, t)2 T 1 =  0  = . and 3PE into the deeper UV. The pulse  ( , )2  2 τ I r t 1 T f length of this laser type is around ∼100 fs I(r, t)dt with a repetition rate of around 100 MHz, T 0 i.e. every 10 ns a short burst of light arrives (3) at the sample while for the remainder Equation 1 then becomes: of this period no light is incident upon P(r)2 1 the sample. Alternative laser sources that n (r)∝δC .(4) F A2 f τ produce similar pulse lengths are based on Cr:LiSrAlFl, Cr:Forsterite, or Nd:YLiF Thus, for example, to obtain the same crystals, or on Yb-doped optical fibers, all of signal with 1 ps pulses rather than with which have much smaller tuning ranges, 100 fs pulses, the average√ power must be but with the exception of the Cr:LiSrAlFl higher by a factor of 10. laser, operate at longer wavelengths than Implicit in this derivation is the can be easily provided by the Ti:Sapphire assumption that the 2P absorption cross laser. A very large tuning range but section δ is constant across the spectrum of less total power is provided by optical the laser pulse. For very short pulses this is parametric amplifiers. not true because – due to the uncertainty An ultrafast laser increases the sig- principle – the shorter the pulse duration nal levels in the 2PM by 5 orders (τ), the broader its spectral width of magnitude as compared to a cw- (ν). For a transform-limited (unchirped) laser of the same average power. This Gaussian-shaped pulse τν = 0.44. A is because the average rate at which 100-fs laser pulse centered around 800 nm fluorescence photons are generated by then ranges (full width at half maximum; 2PA is proportional to the average FWHM) over 10 nm in wavelength and a square of the instantaneous light inten- 20-fs pulse over 50 nm, which is already sity I: as wide as the main features in a typical fluorophore absorption spectrum.  ( )∝δ  ( , )2 nF r C I r t However, in practice the main limitation I(r, t)2 for using shorter pulses is group delay = δCI(r, t)2 ,(1) I(r, t)2 dispersion (GDD), which occurs because Two-photon Microscopy and Imaging 69 different wavelength components of the 2.2 laser pulse travel at different speeds inside Excitation Pathway all materials, including of course, optical glass. After passing the microscope , The pathway for the excitation light in the long-wavelength (‘‘red’’) components a MPM is essentially that of a laser- of the pulse arrive at the focus ahead of the scanning CM: shutter, beam conditioning short-wavelength (‘‘blue’’) components, and intensity control, scan mirrors, scan leading to a so-called chirped pulse. lens, tube lens, objective, and, finally, the Because the wider spectrum of a shorter sample (see Fig. 3). As in every laser- pulse accentuates this effect, an initially scanning microscope, the scan mirrors (or shorter pulse can actually become longer a point in between for proximity coupled once it reaches the focus than an initially scanners) is conjugate to (i.e. imaged into) longer pulse. Owing to the GDD in the objective . Coatings on a typical microscope, a 20-fs pulse, for and mirrors need, of course, to be IR example, is stretched to about 700 fs, while transmissive or reflective, respectively. The a 100-fs pulse is stretched to only 200 fs. intensity control needs to be compatible Without compensation (prechirping, see with ultrashort pulses and should allow below), the shortest pulses that can the automatic compensation of depth be attained at the sample are about dependent losses (see Sect. 3.3) and should 150 fs. Therefore, lasers producing 100 to be fast enough to shut off the beam during 200 fs pulses are typically used in 2PM. retrace, i.e. while the focus is moved from the end of a scan line to the beginning of It is possible (but rarely necessary) to the next. compensate for the microscope’s GDD by the use of an optical arrangement 2.3 (consisting of prisms, gratings, or special Final Focusing and Resolution dielectric coatings) that has negative GDD. Negative GDD prechirps the pulse, Many standard microscope objectives can giving the blue components a head be used for MPM, albeit with compromises start over the red components. This in correction and transmissivity. While head start compensates all or part of thiswasanissueintheearlytimesof the speed disadvantage that the blue MPM, most objectives now are either cor- components have when traveling through rected and transparent throughout the IR the microscope optics. Prechirping allows or come as special ‘‘IR’’ versions. To mini- the arrival of transform-limited pulses as mize tissue damage (see below), detection shortas15fsatthefocus.Forsuchshort efficiency has to be maximized by keep- pulses, compensation of higher-order ing the transmission at the fluorescence dispersion and chromatic aberrations wavelength(s) as high as possible. Optical needs to be considered as well. For higher correction at the fluorescence wavelength than 2PA, the effects of using shorter is not necessary. The numerical aper- pulses are even more dramatic (the 3PA ture (NA) of the objective determines rate, for example, increases by 10 orders of the resolution (laterally ρ ∝ 1/NA,axi- magnitude compared with cw-excitation ally z ∝ 1/(NA)2) and the peak intensity when using the typical Ti:sapphire (∝ (NA)2), and thus the peak excitation laser). rate per fluorophore (∝ (NA)4), but (and 70 Two-photon Microscopy and Imaging

this is a special property of 2PA, that will usually be water or a physiological neither 1P nor higher-order multiphoton salt solution. Refractive-index mismatch absorption, MPA possess) when the con- between immersion medium and sample centration of fluorophores is uniform, the that was not taken into account during the total amount of fluorescence generated lens design results in a loss of resolution throughout the focal volume V = ρ 2z and signal strength. is independent of the NA: 2.4 4 2 NA 2 2 1 Detection n tot ∝ δCP ρ z ∝ δCP . F f τ f τ (5) To preserve the main strength of MPM, i.e. A more extensive treatment that takes the reduction of photodamage, the detec- into account the exact spatial and temporal tion efficiency must not be compromised. intensity distribution of the light in the This is because molecular excitation in- sample yields: evitably causes photodamage to the sample (see Sect. 3.4) and thus the number of 4ηgnλ 2 1 n tot = δCP ,(6) F πh2c2 f τ molecular excitations necessary to gener- ate a sufficient number of detected photons where η is the quantum-efficiency of need to be minimized. In nonscattering the fluorophore (i.e. the percentage of samples, the main determining factor for excited molecules that emit their excess detection efficiency is the solid angle of energy in the form of a photon), g = collection (assuming, of course, the avoid- f τI(r, t)2/I(r, t)2 and depends on the ance of absorption and reflection losses as exact temporal intensity distribution of much as possible). Thus, a high NA objec- the pulse. For the square pulse used tive is crucial if epi detection (through in equation (3), g = 1. For more realistic the objective lens, the usual configura- shapes such as a Gaussian or a hyperbolic tion) is used. Better still is a combination secant intensity distribution, g = 0.66 and of epi detection (where, for working dis- 0.59, respectively; n, λ, h,andc are the tance reasons, a compromise with respect index of refraction, wavelength of the to the NA may be necessary) with trans- excitation light, Planck’s constant, and the detection through a high NA . vacuum speed of light, respectively. The separation of the fluorescence from While scattering per se need not reduce the excitation light by a dichroic mir- theresolutionevenifonlyaminorpart ror is standard practice in fluorescence of the beam energy reaches the focus microscopy, as is the further separation unscattered (ballistically), eventually the into multiple spectral channels. Suppres- stronger attenuation of the lateral rays sion of the excitation light often is easier due to their longer path length inside in MPM because the wavelength sep- thetissuedoesleadtoareductionin aration is larger and the sensitivity of the effective NA, and thus a larger focus some detectors declines sharply toward size.Further,practical,considerationsin longer wavelengths. In contrast to a laser- selecting an objective are the detection scanning CM, there is no need in the efficiency (see below), working distance, MPM to ‘‘de-scan’’ the fluorescence in which tends to decrease with increasing order to thread it through the confocal NA, and the immersion medium, which pinhole. In fact, the detection optics in Two-photon Microscopy and Imaging 71 a MPM can be rather crude but need to 3.1 accept large opening angles and have a Temporal Resolution large field of view for deep imaging in scattering samples. The temporal resolution of optical tech- Deep imaging in scattering samples niques can be very high. In fact, some is where no other known fluorescence of the fastest measurements are based on microscopy technique can compete with pulsed lasers. When fluorescence is used, MPM because MPM can image deep in the excited-state lifetime (in the nanosec- scattering samples virtually without loss of ond range) is the ultimate limit. In practice, resolution and sensitivity as long as the temporal resolution is often limited by the detection pathway collects a large fraction time required to obtain a sufficient signal- of the diffuse fluorescence light emerg- to-noise ratio. When a whole image is to be acquired, this time has to be multi- ing from the sample. Unlike in other plied with the number of pixels in a frame. techniques, the diffuse fluorescence con- Another limitation of all laser-scanning tains high-resolution information about is that the focus has to be the focus because the excitation is highly raster-scanned across the field of view. In localized to a volume of less than one most CMs and MPMs, this is achieved femtoliter. The improvement possible by by mechanically scanning the beam using using whole-field detection has been di- galvanometer mounted mirrors, which are rectly demonstrated by switching quickly limited in frequency response to the low between whole-field and descanned detec- kilohertz regime, setting the rate at which tion. The main drawback of whole-field scan lines can be acquired to about 1 kHz detection is its incompatibility with de- or so, depending somewhat on the ex- tectors that only accept a small phase tent of the scan. A high-resolution (1000 space volume (the product of angular lines) image thus takes at least one sec- and area acceptances). This precludes ond. By sacrificing one spatial dimension the use of photon-counting avalanche and scanning a single line only, a time photodiodes (APDs), which are the high- resolution of 1 ms is common, and when est quantum-efficiency photon detectors measuring only from one spot, a time available, and of spectrometer detectors. resolution in the microsecond range can Fortunately, the characteristics of photo easily be achieved. On the other hand, multiplier tubes (PMTs), which have large to acquire a high-resolution volume im- sensitive areas and acceptance angles age (a stack of optical sections) can take and are the standard detectors for CM, many minutes. When trying to measure have also improved, closing the quantum- dynamic physiological signals from many efficiency gap. locations simultaneously, as it is desirable, for example, in neuroscience, image acqui- sition time is a serious limitation. Attempts 3 have, therefore, been made to speed up the Limitations scanning process by either using resonant scanners, or rotating polygonal mirrors, In this section, the limitation of spatial and or by scanning the beam nonmechanically temporal resolution, penetration depth using an acousto-optical deflector (AOD). and photodamage will be discussed. Owing to the spread wavelength spectrum 72 Two-photon Microscopy and Imaging

of the excitation pulses, the AOD, which point-spread function) is completely de- is based on diffraction by a sound wave, is termined by the intensity distribution (in plagued by angular dispersion, which can fact, the point-spread function equals the only be sufficiently compensated for a lim- intensity squared) at the focus. Thus, the ited field of view. The main attraction of resolution depends on the wavelength of AOD scanning is the capability of random- the excitation light and on the NA of the access scanning: the focus can be moved objective. With 700-nm light and an NA of between distant location within microsec- 1.3, a resolution (FWHM) of ∼200 nm in onds without illuminating (and potentially the lateral and 600 nm in the axial direction damaging) the sample area in between. can be achieved. Acquisition speed is limited ultimately, Even though in CM the resolution is as mentioned above, by the fluorescence determined by the combined (multiplied) decay time, which has to be shorter than excitation and detection point-spread func- the pixel dwell time. But the limited tions, the mathematical expression for the rate at which a fluorophore can produce resolution of CM and 2PM are identi- photons, even if the excitation light is cal. This holds, however, only under the arbitrarily intense, usually enforces a somewhat unrealistic assumption that the longer pixel dwell time just to collect pinhole has zero diameter and the 1PE enough photons. Beyond those limits, and 2PE wavelengths and the emission an increase is only possible by scanning wavelength are all the same. Realistically, multiple points, arranged either in a 2D the pinhole has to be opened up to gain pattern or in a line. This approach is sufficient signal, the emission wavelength rather successful in CM, but it requires is longer (both reducing the resolution of confocal or at least spatially resolved CM) and the excitation wavelength is close detection, since at each point in time, to twice as long for 2P as for1P (reducing fluorescence can come from any of the the resolution in 2PM). foci. Thus, using it in MPM means As a result, compared with CM, the giving up one of the main advantages resolution in 2PM (and MPM, in general) of MPM, namely the ability to detect is typically somewhat worse, but can be fluorescence irrespective of how it reaches improved by reintroducing a confocal the detector (see above) and accepting pinhole, albeit at the expense (particularly either image degradation or signal loss. in scattering specimens) of detection Other drawbacks are anisotropic lateral efficiency. 2PM can also be combined and reduced axial resolution (for line with the , which greatly illumination) and the increased laser- improves the resolution in the axial power requirement (for any multipoint direction, and where using 2PE rather than method). 1PE considerably reduces side lobes in the point-spread function and thus fills in the 3.2 holes in the modulation transfer function. Spatial Resolution In general, at a given wavelength, res- olution improvements beyond the Abbe In the generic configuration (with whole- limit require optical nonlinearity, which field detection), there is no spatial se- is exploited in the Stimulated Emission lectivity in the detection and hence the Depletion (STED) microscope. This mi- resolution of MPM (i.e. the size of the croscope is based on the saturation of Two-photon Microscopy and Imaging 73 excited-state depletion, which has poten- To increase the imaging depth further, tially unlimited resolution, because this the efficiency in generating 2P fluores- saturation contains arbitrarily high or- cence needs to be increased, which is possi- ders of nonlinearity. 2PE, by contrast, ble by reducing the laser pulse–repetition contains only a second order (quadratic) rate while increasing the pulse energy. nonlinearity, and consequently, in 2PM The use of a so-called regenerative amplifier, the resolution improvement beyond the which generates pulses that are about 250 Abbe limit is moderate. times as intense as those directly from the laser ‘‘oscillator’’ but are also by the same 3.3 factor less frequent, resulting in an un- Penetration Depth changed average power, permits imaging to about 1000 µm. A somewhat techni- In scattering samples where absorption calbutimportantissueisthateachpixel of the light is negligible, the intensity of needs to contain at least one pulse, limiting the ballistic light, i.e. light that has not the pixel rate to several hundred kilohertz been scattered, decreases with depth in (the repetition rate of the amplifier). At the sample as: such pulse rates, the average 2PA rate is   d increased by a factor of about 250. I = I0 exp − ,(7) Now the imaging depth is, however, ball l s no longer limited by the available laser where ls is the scattering length, i.e. power but instead by the fluorescence the average distance a photon travels background generated near the surface of before it is scattered. For near-infrared the sample. The reason for this is that wavelengths, ls in brain tissue is around in order to keep the fluorescence gener- 200 µm. To estimate the imaging depth, ation at the focus constant, the amount one can assume that only the ballistic of ballistic light that reaches the focus lightthatreachesthefocuscontributesto must also be kept the same, and thus the the generation of two-photon fluorescence, laser power that needs to enter the sample which seems to hold for up to at least must increase exponentially (equation 7). several scattering mean-free-path lengths. At large depths, this exponential increase Then the number of fluorescence photons overcomes the quadratic decrease in light generated when the focus is at a depth d intensity, which results from the increased below the surface can be written as: beam cross section with distance from the 1 g 8nλ focus. Eventually, the intensity at the sur- 2 (−2d/ls) nFtot = ηδCP e .(8) face becomes comparable to the intensity 2 f τ πh2c2 at the focus and surface fluorescence starts As long as the detection system is de- to dominate. For samples with staining signed to capture most of the fluorescence, throughout the sample, this limit may well which consists almost entirely of scattered be impossible to overcome, thus defining light, the maximum imaging depth de- the ultimate depth limit of 2PM (a similar pends only on the laser power that is logic applies to higher-order MPM). This available. With the power available from limit increases somewhat with the NA, but standard mode-locked Ti:sapphire lasers, that may not help much since at higher imaging depths of 2 to 3 scattering lengths NAs, focus blurring due to wavefront aber- (400–600 µm) are achieved routinely. rations becomes a more serious issue, and 74 Two-photon Microscopy and Imaging

may, in turn, require the use of adaptive nonlinearities such as locally overwhelm- optics as a remedy. ing cellular repair mechanisms should be avoidable by faster scanning. Often, 3.4 as mostly anecdotal evidence suggests, Photodamage and Photobleaching damage decreases with increasing wave- length, which may be due to reduced Cell viability is obviously important for 2PA by endogenous chromophores. While imaging living tissue. Excitation of in- photobleaching can be quantitatively mea- trinsic or introduced chromophores often sured, this is more difficult with damage leads to photochemical effects, such as to cells, since photostress may be present destruction of the chromophore (bleach- but may not be evident below a certain ing) or damage and subsequent cell-death, instantaneous or cumulative threshold. which are mostly mediated by reactive This is consistent with the observation oxygen species. This problem is exacer- in several studies that at low intensities bated in laser-scanning CM because of the (up to 1014 − 1015 Wm−2 which corre- rather poor utilization of the generated sponds to 1–5 mW at the focus) no fluorescence light and the appearance of obvious damage occurs. As the intensity superlinear photochemical effects at high increases, severe damage, optical break- intensities, probably due to excited-state down, and strong luminescence eventually absorption. Utilization of the generated do occur. fluorescence is generally much better in MPM, with the improvement increasing as the specimen becomes more scattering 4 and the imaging depth increases. Fluorophores However, in clear (i.e. nonscattering) specimens, where the detection efficiency 4.1 in the CM for focal photons can be rather Comparison of 1PA and 2PA high, bleaching and photodamage in the 2PM can actually be exacerbated, but is While the fluorescence process itself is also clearly reduced in some cases com- largely independent of the mode of excita- pared with CM. Strategies for avoiding or tion (and hence the fluorescence emission reducing damage depend on the under- spectra are the same for 1PE and 2PE), lying photophysics and photochemistry. the absorption spectra of 1PE and 2PE are If damage is due to single-photon ab- expected and found to be quite different in sorption, increasing the 2PE efficiency by some cases. The reasons for this are: (1) To reducing pulse length or repetition rate excite a molecule from its ground state to is helpful. Damage is, however, rarely the excited state, quantum-mechanical se- dominated by 1PA for typical tissue, but lection rules have to be fulfilled in addition can become so in pigmented cells or to energy conservation. For example, the if much longer wavelengths are used angular momentum (usually only the pho- and water absorption becomes an issue. ton spin is relevant since the absorption If higher-order instantaneous nonlinear- of a photon with nonzero orbital angular ities, such as 3P absorption or optical momentum is unlikely) that is carried by a breakdown are the culprits, increasing photon must be taken up by the molecule the pulse length can help. Chemical upon absorption. The selection rules for Two-photon Microscopy and Imaging 75

2PA are different from those for 1PA, since attempts to predict 2P cross sections and in the first case, two photons, each carrying to develop, on the basis of theoretical aspinof1¯h, are absorbed. This means that calculations, chromophores with large 2P the angular momentum of the molecule cross sections. has to be unchanged (if two photons of In the following, we will give a short opposite spin are absorbed) or changed by overview of chromophores that are widely 2¯h.Achangeby1¯h (also corresponding to used with 2PM, with particular focus a change in parity) is not allowed. How- on dyes with biological relevance. Under ever, for complex asymmetric molecules, this premise, the chromophores can be these selection rules are not necessarily categorized into intrinsic, synthetic, and as strict, due to interaction with molecu- genetically encoded dyes, each group lar vibrations and rotations. (2) Since the containing dyes with a variety of different transition to the excited state occurs in a indicator features. sense via all molecular states, which serve as a combined ‘‘virtual’’ intermediate state, 4.2 excitation is possible even if there is no di- Intrinsic Chromophores rect orbital overlap between ground and target state. Biological tissue is naturally fluorescent For the use of fluorescent dyes in to a varying degree even without adding MPM, it is very important to know chemically synthesized molecules or in- their MP-absorption spectra in order to troducing the gene of a fluorescent choose the best dye and then excite (see below). The molecular origin of this it optimally. While it is not easy to intrinsic ‘‘autofluorescence’’ depends on measure MP-absorption spectra, at least the excitation wavelength. For UV excita- the 2P-absorption spectra of many com- tion, nucleotides and the aromatic amino monly used chromophores are now avail- acids are dominant, for excitation with visi- able (http://www.drbio.cornell.edu/) and ble wavelengths, other compounds such as it turns out that all dyes used with 1PE can nicotinamide adenine dinucleotide (phos- also be used with 2PE, albeit with a larger phate) NAD(P)H and flavin adenine dinu- spread in their cross sections because, in cleotide FAD are the main contributors. a way, transition matrix elements enter twice in the 2PA process. Tryptophane fluorescence is rarely used In some cases, the 2PA spectra plotted for 2PM, but 2PE fluorescence correlation on a λ/2 scale are rather similar to spectroscopy (FCS) is possible for the 1PA spectra plotted on a λ scale. containing a large number of tryptophane In most cases, however, the excitation residues. NAD(P)H is of special interest maxima are shifted to the blue and the as its fluorescence intensity depends spectra are broader. Additional spectral on its oxidation state. With excitation features at wavelengths longer than the at 800 nm, NAD(P)H and flavoprotein ‘‘red’’ 1P absorption edge are not seen, fluorescence can be used together for a nor would they be expected. The reason quantitative ratiometric measurement of for this is that fluorescence emission only cellular metabolism. occurs for molecules where the transition One of the advantages of MPA for to the lowest lying excited state 1P is exciting cellular autofluorescence is that allowed. There have been a number of it permits access to transitions that 76 Two-photon Microscopy and Imaging

require for 1PE, ultraviolet light of wave- moderate 2P cross section. Much larger lengths that do not pass most micro- cross sections are found in xanthene- scope objectives. derived indicators, which also use the + On one hand, endogenous fluorophores BAPTA Ca2 -binding group and are now + can be useful because tissues can be im- available with a wide range of Ca2 aged without dyes having to be applied affinities, covering the entire physiological to the tissue. On the other hand, endoge- range. A selection of different peak emis- nous fluorophores contribute most of the sion wavelengths (from 530 to 670 nm) unwanted background when exogenous ensures discrimination ability against GFP dyes, which provide enormous specificity or other cellular labels if needed. and tailored indicator properties, are to be In tissue imaging, labeling without imaged. A special case is the green fluo- damage to cells is crucial and several rescent protein (GFP, see below), which methods have been used in connection occurs naturally in the jellyfish and has with MPM. (1) Loading individual cells a biological function there, but in other by diffusion or iontophoresis from dye- organisms GFP is an exogenous stain in filled micropipettes in brain slices or the sense that it needs to be introduced by in vivo; (2) applying cell-permeable ace- molecular genetic means. toxymethoxyl (AM) esters variants, which become trapped in cells by intracellular es- 4.3 terases. AM loading is straightforward in Synthetic Dyes isolated cells, but is also possible in brain slices and even in vivo;and(3)particle Because of the specific advantage of (‘‘gene’’)-gun delivery. MPM for the observation of living tissue, For an overview of fluorescent indicators the most frequently used synthetic dyes see http://www.probes.com/. All of these are those that provide functional signals, indicators can be efficiently 2P excited called fluorescent indicators,inparticular, somewhere between 700 and 1000 nm, ion-sensitive, and (more recently) voltage- i.e. they are accessible when using a sensitive probes. Ti:sapphire laser.

4.3.1 Ion Indicators 4.3.2 Voltage-sensitive Dyes In many cellular responses, changes in ion Voltage-sensitive dyes (VSDs) measure concentrations are involved. Most impor- electrical activity directly rather than in- 2+ + tant is Ca , with its key position in cellular directly via Ca2 influx. Most VSD-based signal transduction; also important are measurements have, until recently, not + + 2+ + − Na ,K ,Mg ,H ,andCl .Ionin- used laser-scanning microscopy, since the dicators go back to Arnaldus de Villanova, fractional fluorescence changes that oc- adoctorandalchemist(∼1300 AD), who cur with physiological voltage swings are invented litmus, an indicator that changes small and thus need for detection and its color (absorption spectrum) depend- quantification of a large number of fluores- + ing on the concentration of H ions. The cent photons. Recently, it was shown that first 2PM calcium measurements where by using band-edge excitation, fractional + done using Indo-1, a BAPTA-based Ca2 changes are substantially increased, and indicator, which is, however, excited by are for equivalent excitation wavelengths, rather short wavelength and has a rather larger with 2PE than with 1PE. Two-photon Microscopy and Imaging 77

4.4 only method that allows morphological Genetically Encoded Fluorophores and and functional imaging in thick living Indicators tissue and in live animals. While many of the essential uses of MPM are in Labeling with synthetic dyes is in most the field of neuroscience, the range of cases not cell-type specific. For example, biological structures and processes in- + when injecting AM ester-Ca2 indicator vestigated using MPM have continuously into brain tissue, many cell types take up grown. Applications of MPM now include the dye, and it is impossible to stain, for ex- photodynamic therapy, noninvasive opti- ample, only neurons. Genetically encoded cal biopsy of human skin, the study of protein ‘‘dyes,’’ often called XFPs by gen- cellular metabolism, embryogenesis, im- eralizing from GFP, the green fluorescent munology, tumor pathophysiology, and protein, with ‘‘X’’ standing for the color neurodegenerative disease. In the fol- such as for yellow in YFP and for cyan lowing, we will discuss some of these in CFP, overcome this problem because applications in more detail. their expression can be put under the con- trol of specific promoters that are only 5.1 active in certain cell types. XFPs can also Neurobiology be fused to other proteins to then map their dynamic distribution in the cell. Fu- After it became clear that 2PM is par- sion protein with XFPs have also been ticularly well suited to high-resolution designed to act as indicators for Ca2+, − imaging in scattering tissue, it was ap- Cl , pH, and other cellular signals and plied to neural tissue, which is strongly physiological parameters such as cAMP, scattering and requires the use of in- phosphorylation, redox potential, protein tact pieces of tissue for functional studies kinase B and C, as well as membrane volt- owing to the high degree of cellular in- age. The molecular genetic methods used terconnectivity. Early studies focused on + to label intact animals are either the cre- Ca2 dynamics in dendritic spines. Then ation of transgenic animals, with it became clear that it is possible to im- + properly engineered viral vectors, or in vivo age Ca2 dynamics with high spatial and electroporation. temporal resolution in the wholly intact For imaging in intact animals XFPs brainaswell,where2PMcannowbe and MPM are rather synergistic, since used to guide recording pipettes to se- together they solve the deep labeling and lected cells. It has also been possible to the deep imaging problem, with 2PM even study the structural stability and plastic- allowing imaging through the thinned but ity of neuronal morphology in explanted intact skull. tissue and, over periods of months, in intact animals. 2PM has helped to reveal neural struc- 5 ture and function at various levels ranging Applications from individual synapses to entire neural networks (see also Fig. 4). The retina, a Onthebasisoftheuniqueimaging spatially separate but functionally integral properties described earlier, MPM has part of the brain, has been a particularly re- become the preferred and virtually the warding specimen for 2PM. Because of the 78 Two-photon Microscopy and Imaging

0 0

200 200

621 µm 621 µm 400 400

600 860 µm 600 860 µm

800 800 1028 µm 1028 µm

1000 1000

µ µ (a) 50 m (b) 50 m

ds dt ∆t/∆s ∆t ∆s

900 µm 300 ms

25 µm (c) Fig. 4 (a) x-z projection and single planar scans of GFP labeled neurons and (b) stained vasculature obtained throughout almost the entire gray matter of the mouse neocortex. (c) Blood flow measurement in the mouse neocortex at 900 µm below the brain surface. Blood cells appear as shadows in surrounding blood plasma. Their motion traces out shaded bands in an image consisting of line scans repeatedly taken along the capillaries (dashed line in the planar scan). Blood flow parameters as velocity, linear density, average spacing, and flux of red blood cells can be inferred from the slope (dt/ds) of, and the distance (ds)andtime(dt) between shaded bands.

retina’s high sensitivity for UV and visible in fact, intensities used commonly for flu- light, 1PE of common fluorescent probes orescence microscopy completely bleach inevitably perturbs the observed specimen; the photopigments within seconds. In Two-photon Microscopy and Imaging 79 contrast, photoreceptors are blind to light muscular contraction and neurotransmit- in the near-infrared wavelength region, ter secretion, and because [Ca2+]tran- allowing the use of IR-excited 2PM to mea- sients can often be used as a proxy for sure light stimulus-evoked calcium signal electrical excitation. The particular advan- in the functionally intact retina, which has tages of MPM are, again, excitation-based been used to study the mechanism of mo- optical sectioning and highly efficient flu- tion detection in retinal cells (see Fig. 5). orescence collection in scattering tissue. A disadvantage of MPM is that ratiomet- 5.2 ric indicators that require dual-wavelength Calcium Imaging excitation are difficult to use because a second laser is required for the second ‘‘Calcium imaging’’ constitutes the bulk excitation wavelength (at this point in of ion imaging, both with 1PM and with time, changing the wavelength even in MPM, because of the important role of a fully automated Ti:Sapphire lasers still + Ca2 in coupling electrical processes to takes seconds). Therefore, ratiometric cal- biochemical processes, for example, in cium imaging with 2PM has to resort

50 µm

(a) Fig. 5 Two-photon optophysiology in the retina. Dye-filled ‘‘starburst’’ amacrine cell (a) in flat-mounted rabbit retina. Like many amacrine cells, this neuron bears no axon; it receives inputs and makes output synapses with its dendrites. Starburst cells are involved in the detection of image motion. Using (b) two-photon microscopy, light stimulus-evoked Ca2+ signals (green trace) were recorded in the dendritic tips of a starburst amacrine cell. Simultaneously, membrane voltage (black trace) was measured at the soma using a patch electrode (see schematic drawing). The light stimulus, a concentric sinusoidal wave, induced stronger responses when it was expanding (left) than when it was contracting (right) (see color plate p. xxv). 80 Two-photon Microscopy and Imaging

‘Expansion’ ‘Contraction’

40% 2+ [Ca ] ∆F/F

Vm 1 mV

0.5 s (b) Fig. 5 (Continued)

to Indo-1, which has drawbacks, such as the activity of respiratory enzymes and a short absorption wavelength and fast thus is an intrinsic probe of cellular bleaching, but has been used to mea- metabolism. 1P-CM of two-dimensional sure calcium oscillations in tumor mast NADH/NADPH-fluorescence maps is pos- cells and calcium transients in cardiac my- sible, as has been shown in rabbit cornea, ocytes. More common nowadays is the use but is hampered by photobleaching and of dye mixtures that provide virtually all photodamage due to the UV illumina- the advantages of proper ratiometric in- tion (∼360 nm) needed for the excitation dicators. Furthermore, single-wavelength of NADH/NADPH. Those problems are indicators are often sufficient, particularly substantially reduced with 2PE so that if one is interested mainly in the temporal now nearly continuous NADH/NADPH + [Ca2 ] dynamics (see Figs. 5 and 6). Satis- imaging is possible throughout the entire factory calibration can often be achieved by thickness (400 µm) of the rabbit cornea. using the intensity prior to stimulation as Because metabolic events can be fast but a reference, such as was done in the early may need to be monitored for extended + studies of [Ca2 ] dynamics in dendrites, time periods, low-damage imaging is of and dendritic spines in brain slices, and in paramount importance. With MPM, it has whole animals. become possible to follow the response to glucose application in β-cells inside intact 5.3 pancreatic islets and in muscle cells. Imaging of Metabolic Activity 5.4 The fluorescence from the reduced pyri- Photoactivation (Uncaging) dine nucleotides NADH and NADPH, which are metabolic intermediates, can The excellent localization of MPE can be be used to localize and characterize used to confine photochemical generation Two-photon Microscopy and Imaging 81

(a) 50 µm (b) 5 µm Fig. 6 Measurement of calcium dynamics in dendritic spines of a hippocampal neuron (a) using simultaneous excitation, at 910 nm, of two different fluorophores – a red fluorescent Ca2+-insensitive dye (Alexa-594) helping in visualization of small structures and a green fluorescent Ca2+ indicator (Fluo-5F), which is very dim at rest, but shows large fluorescence changes in activated spines. (b) Following extracellular electric stimulation, presynaptic fibers release transmitter in the synaptic cleft whose binding to corresponding receptors on the postsynaptic membrane causes a Ca2+ rise in a single dendritic spine leading to an increase in green fluorescence observed by successive planar scans of a dendritic branchlet (see color plate p. xxvi). of chemical compounds, such as flu- combination of 2P uncaging and bleaching orescent tracers or biological signaling of fluorescence has been used to measure molecules, to volumes smaller than a fem- diffusional resistance of spine necks. toliter. Typically, this is achieved by the illumination of a photolabile precursor 5.5 that then absorbs two or more photons Human Diseases and Therapy and decays via a photochemical pathway into the desired product. Scanning 2P pho- In conjunction with animal models, MPM tochemical microscopy, first demonstrated has provided some insights into human by mapping the distribution of neurotrans- diseases such as Alzheimer’s demen- mitter receptors on cultured muscle-like tia. There, 2PM in vivo imaging allows cells, has, however, been hampered by the the observation over time of how senile very low 2PE cross sections of the available plaques (amyloid-beta peptide aggregates) ‘‘caged’’ compounds (an example of the develop. This is extremely helpful when rule that weak 1P absorbers tend to be very evaluating antiplaque therapies, since the weak 2P absorbers) even though there has fate of individual plaques can be fol- been recent progress in the synthesis of lowed over weeks and months by repeated better caged compounds, having allowed imaging through a thinned skull. These the study of calcium ‘‘sparks’’ in cardiac experiments have shown that, surpris- muscle cells and the mapping of gluta- ingly, plaques do not change size af- mate sensitivities on dendritic spines. A ter formation. 82 Two-photon Microscopy and Imaging

2PM has also been used to measure MPM, the range of applications that bene- blood flow at the level of single capillar- fit from MPM continues to expand. Much ies and to observe leucocytes-endothelial of the current improvement efforts are interactions in tumors in vivo at tissue directed at excitation and detection effi- depths that are not accessible otherwise. ciencies, which, as was discussed at length Such studies have revealed, for example, earlier, is the main factor determining the depth dependent dynamics of tumor imaging depth and specific (i.e. per in- angiogenesis. In the area of immunology, formation gained) photodamage. Novel 2PM allows the observation of lymphocytes chemical fluorophores with large two pho- in their native environment such as mon- ton–absorption cross sections still await itoring the motility of T cells that invade significant biological applications. Closer the brain during neuroinflammation. to being really useful appear to be semicon- Potential clinical applications on MPM ductor quantum dots (QDs), which have are optical skin biopsy and photodynamic the largest 2PA cross sections measured, therapy. Traditional biopsy is an invasive are very photostable, and are available with procedure, which requires the removal and widely varying but narrow emission spec- tra. Recent advances have all but solved fixation of tissue before imaging. During early problems with solubility, quenching, this process, biochemical information is and toxicity. Different coats allow covalent often poorly preserved. Optical biopsy does linking to biorecognition molecules, such not require the removal of any tissue as or biotin/avidin, so that QDs sample, but it remains to be seen whether can be used as specific fluorescent probes. the quality of 2PM images will become In conjunction with their broad excitation good enough to allow a pathological spectra, QDs are very well suited for mul- analysis with accuracy comparable to that tilabel imaging. of traditional biopsies. An entirely different route to improving Photodynamic therapy is based on the excitation efficiency is the use of coherent preferential accumulation of certain pho- control, which involves tailoring the phase tosensitizing agents in tumorous or other- and amplitude of a laser pulse to optimize wise abnormal tissues and the subsequent the optical response of a molecule, to selective tissue destruction by illumina- achieve selective excitation, or to reduce tion. 2PE photodynamic therapy has the photobleaching. potential of providing more spatially spe- Inhomogeneities in the index of refrac- cific destruction of, for example, cancer tion across the tissue lead to degradation tissue than 1PE, which can be confined of the focus and thus a reduction in 2PE not as easily or not at all. efficiency. In particular at large depths, therefore, adaptive aberration correction can significantly improve excitation. 6 In order to image structures that are Future Directions beyond the penetration depth of the MPM, overlying tissue has to be removed. In Among the ‘‘exponentially’’ growing num- order to keep the lateral extent of such ber of papers on MPM, about half are removal, limited ‘‘endoscopic’’ approaches still on methods development. With these are being developed that use a small ongoing improvements and extensions of diameter gradient-index (GRIN) lens as Two-photon Microscopy and Imaging 83 the objective. This limits the NA and the Denk, W., Piston, D.W., Webb, W.W. (1995) field of view, but may be the only way to Two-photon molecular excitation in laser reach deeper brain structures such as the scanning microscopy, in: Pawley, J. (Ed.) The Handbook of Confocal Microscopy, Plenum, hippocampus in vivo. New York, pp. 445–458. Regular MP microscopes are bulky and Diaspro, A., 2002 Confocal and Two-photon thus most in vivo imaging is done in Microscopy: Foundations, Applications, and anesthetized and immobilized animals. To Advances, Wiley-Liss, New York, pp. xix, achieve the goal of high-resolution imag- 567. Helmchen, F., Denk, W. (2002) New develop- ing in freely moving animals, scanners ments in multiphoton microscopy, Curr. Opin. and optics have to be miniaturized. This Neurobiol. 12(5), 593–601. requires a different scanning mechanism Konig,¨ K. (2000) Multiphoton microscopy in life because the weight of a galvo scanner rules sciences, J. Microsc. (Oxford) 200, 83–104. out its use in a headpiece that can weigh at Masters, B.R., Thompson, B.D. (Eds.) (2003) most a few tens of grams without encum- Selected Papers on Multi-photon Excitation Microscopy, SPIE Milestone Series, Vol. bering an animal such as a rat too much. MS175. SPIE Press, Bellingham, D.C. An alternative is the ‘‘2P-fiberscope’’ in Mertz, J. (2004) Nonlinear microscopy: new which scanning is achieved by deflecting techniques and applications, Curr. Opin. thefreeendofthefiberthatisusedto Neurobiol. 14(5), 610–616. deliver the excitation light to the head- So, P.T.C., et al. (2000) Two-photon excitation piece. The deflection can, for example, fluorescence microscopy, Annu. Rev. Biomed. Eng. 2, 399–429. be achieved by using resonant oscilla- Zipfel, W.R., Williams, R.M., Webb, W.W. tions. One challenge peculiar to nonlinear (2003) Nonlinear magic: multiphoton fiber microscopy is that special measures, microscopy in the biosciences, Nat. Biotechnol. including the use of large core or mi- 21(11), 1368–1376. crostructured fibers, have to be taken to limit the degradation of the pulse shape Primary Literature by nonlinear optical effects in the beam- delivery fiber. Abbe, E. (1873) Beitrage¨ zur Theorie des Mikroskops und der mikroskopischen Wahrnehmung, Schultzes Arch. Mikrosk. Anat. 9, 413–468. See also Alzheimer’s Disease; Cal- Albota, M., et al. (1998) Design of organic molecules with large two-photon absorption cium Biochemistry; Metabolic Ba- cross sections, Science 281(5383), 1653–1656. sis of Cellular Energy; Molecular Barad, Y., et al. (1997) Nonlinear scanning laser microscopy by third harmonic generation, Neurobiology, Single-Cell; Neuron Appl. Phys. Lett. 70(8), 922–924. Chemistry. Bardeen, C.J., et al. (1999) Effect of pulse shape on the efficiency of multiphoton processes: implications for biological microscopy, J. Bibliography Biomed. Opt. 4(3), 362–367. Beaurepaire, E., Mertz, J. (2002) Epifluorescence collection in two-photon microscopy, Appl. Books and Reviews Opt. 41(25), 5376–5382. Beaurepaire, E., Oheim, M., Mertz, J. (2001) Denk, W., Svoboda, K. (1997) Photon upman- Ultra-deep two-photon fluorescence excitation ship: why multiphoton imaging is more than in turbid media, Opt. Commun. 188(1–4), a gimmick, Neuron 18(3), 351–357. 25–29. 84 Two-photon Microscopy and Imaging

Bennett, B.D., et al. (1996) Quantitative sub- de Grauw, G.J., et al. (1999) Imaging properties cellular imaging of glucose metabolism within in two-photon excitation microscopy and intact pancreatic islets, J. Biol. Chem. 271(7), effects of refractive-index mismatch in thick 3647–3651. specimens, Appl. Opt. 38(28), 5995–6003. Bewersdorf, J., Pick, R., Hell, S.W. (1998) Denk, W. (1994) 2-photon scanning photo- Multifocal multiphoton microscopy, Opt. Lett. chemical microscopy-mapping ligand-gated 23(9), 655–657. ion-channel distributions, Proc. Natl. Acad. Sci. Bhawalkar, J.D., et al. (1997) Two-photon U.S.A. 91(14), 6629–6633. photodynamic therapy, J. Clin. Laser Med. Surg. Denk, W., et al. (1994) Anatomical and 15, 201–204. functional imaging of neurons using 2-photon Bird, D., Gu, M. (2003) Two-photon fluorescence laser-scanning microscopy, J. Neurosci. endoscopy with a micro-optic scanning head, Methods 54(2), 151–162. Opt. Lett. 28(17), 1552–1554. Denk, W., Detwiler, P.B. (1999) Optical Birge, R.R. (1986) 2-photon spectroscopy of recording of light-evoked calcium signals in protein-bound chromophores, Acc. Chem. Res. the functionally intact retina, Proc. Natl. Acad. 19(5), 138–146. Sci. U.S.A. 96(12), 7035–7040. Brakenhoff, G.J., et al. (1996) Real-time two- Denk, W., Strickler, J.H., Webb, W.W. (1990) photon confocal microscopy using a Two-photon laser scanning fluorescence femtosecond, amplified Ti:sapphire system, J. microscopy, Science 248, 73–76. Microsc. (Oxford) 181, 253–259. Denk, W., Sugimori, M., Llinas, R. (1995) Two Brown, E.B., et al. (2001) In vivo measurement types of calcium response limited to single of , angiogenesis and spines in cerebellar Purkinje cells, Proc. Natl. physiological function in tumors using Acad. Sci. U.S.A. 92(18), 8279–8282. multiphoton laser scanning microscopy, Nat. + Eilers, J., et al. (2001) GABA-mediated Ca2 Med. 7(7), 866–870. signaling in developing rat cerebellar Purkinje Centonze, V.E., White, J.G. (1998) Multiphoton neurones, J. Physiol. (London) 536(2), excitation provides optical sections from 429–437. deeper within scattering specimens than Engert, F., Bonhoeffer, T. (1999) Dendritic spine confocal imaging, Biophys. J. 75(4), changes associated with hippocampal long- 2015–2024. term synaptic plasticity, Nature 399(6731), Chalfie, M., et al. (1994) Green fluorescent 66–70. protein as a marker for gene expression, Science 263(5148), 802–805. Euler, T., Detwiler, P.B., Denk, W. (2002) Chan, W.C.W., Nie, S.M. (1998) Quantum dot Directionally selective calcium signals in bioconjugates for ultrasensitive nonisotopic dendrites of starburst amacrine cells, Nature detection, Science 281(5385), 2016–2018. 418(6900), 845–852. Chance, B., Thorell, B. (1959) Localization and Fan, G.Y., et al. (1999) Video-rate scanning two- kinetics of reduced pyridine nucleotide in photon excitation fluorescence microscopy living cells by microfluorometry, J. Biol. Chem. and ratio imaging with cameleons, Biophys. 234(11), 3044–3050. J. 76(5), 2412–2420. Christie, R.H., et al. (2001) Growth arrest of Feierabend, M., Ruckel, M., Denk, W. (2004) individual senile plaques in a model of Coherence-gated wave-front sensing in Alzheimer’s disease observed by in vivo strongly scattering samples, Opt. Lett. 29(19), multiphoton microscopy, J. Neurosci. 21(3), 2255–2257. 858–864. Fork, R.L., Martinez, O.E., Gordon, J.P. (1984) Chung, M.A., Lee, K.S., Jung, S.D. Two- Negative dispersion using pairs of prisms, Opt. photon absorption cross sections of Lett. 9(5), 150–152. dithienothiophene-based molecules, ETRI J. Friedrich, D.M. (1982) 2-photon molecular- 2002. 24(3), 221–225. spectroscopy, J. Chem. Educ. 59(6), 472–481. Cohen, L.B., et al. (1974) Changes in axon Gannaway, J.N., Sheppard, C.J.R. (1978) Second- fluorescence during activity: molecular probes harmonic imaging in the scanning optical of membrane potential, J. Membr. Biol. 19(1), microscope, Opt. Quantum . 10(5), 1–36. 435–439. Two-photon Microscopy and Imaging 85

Goeppert-Mayer, M. (1931) Ueber Elemen- mammalian cells, Proc.Natl.Acad.Sci.U.S.A. tarakte mit zwei Quantenspruengen, Ann. 96(11), 6255–6260. Phys. 9, 273. Jovin, T.M. 2003 Quantum dots finally come of Griesbeck, O. (2004) Fluorescent proteins as age, Nat. Biotechnol. 21(1), 32–33. sensors for cellular functions, Curr. Opin. Jung, J.C., Schnitzer, M.J. (2003) Multiphoton Neurobiol. 14(5), 636–641. endoscopy, Opt. Lett. 28(11), 902–904. Grutzendler, J., Kasthuri, N., Gan, W.B. (2002) Kaiser, W., Garrett, C.B.G. (1961) Two-photon 2+ Long-term dendritic spine stability in the adult excitation in CaF2:Eu , Phys. Rev. Lett. 7(6), cortex, Nature 420(6917), 812–816. 229–231. Gu, M. (1996) Resolution in 3-photon Kawano, H., et al. (2003) Attenuation of fluorescence scanning microscopy, Opt. Lett. photobleaching in two-photon excitation 21(13), 988–990. fluorescence from green fluorescent protein Gu, M., Sheppard, C.J.R. (1995) Comparison of with shaped excitation pulses, Biochem. 3-dimensional imaging properties between Biophys. Res. Commun. 311(3), 592–596. 2-photon and single-photon fluorescence Kettunen, P., et al. (2002) Imaging calcium microscopy, J. Microsc. (Oxford) 177, 128–137. dynamics in the nervous system by means Hanninen, P., Soini, E., Hell, S. (1994) Contin- of ballistic delivery of indicators, J. Neurosci. uous wave excitation two-photon fluorescence Methods 119(1), 37–43. microscopy, J. Microsc. (Oxford) 176, 222–225. Kim, K.H., Buehler, C., So, P.T.C. (1999) High- Hell, S., Stelzer, E.H.K. (1992) Fundamental speed, two-photon scanning microscope, Appl. improvement of resolution with a 4pi- Opt. 38(28), 6004–6009. confocal fluorescence microscope using 2- Kleinfeld, D., et al. (1998) Fluctuations and photon excitation, Opt. Commun. 93(5–6), stimulus-induced changes in blood flow 277–282. observed in individual capillaries in layers 2 Hell, S., Wichmann, J. (1994) Breaking the through 4 of rat neocortex, Proc. Natl. Acad. diffraction resolution limit by stimulated Sci. U.S.A. 95(26), 15741–15746. emission: stimulated emission depletion Koester, H.J., et al. (1999) Ca2+ fluorescence fluorescence microscopy, Opt. Lett. 19(11), imaging with pico- and femtosecond two- 780–782. photon excitation: signal and photodamage, Hell, S.W., Dyba, M., Jakobs, S. (2004) Concepts Biophys. J. 77(4), 2226–2236. for nanoscale resolution in fluorescence Konig,¨ K., Tirlapur, U.K. (2002) Cellular and microscopy, Curr. Opin. Neurobiol. 14(5), 599–609. Subcellular Perturbations during Multiphoton Hellwarth, R., Christiansen, P. (1974) Nonlinear Microscopy, in: Diaspro, A. (Ed.) Confocal optical microscopy examination of structure and Two-photon Microscopy. Foundations, in polycrystalline ZnSe, Opt. Commun. 12(3), Applications and Advances, Wiley-Liss, New 318–322. York, pp. 191–205. Helmchen, F., et al. (1999) In vivo dendritic Kuhn, B., Fromherz, P., Denk, W. (2004) High calcium dynamics in deep-layer cortical sensitivity of stark-shift voltage-sensing dyes pyramidal neurons, Nat. Neurosci. 2(11), by one- or two-photon excitation near the red 989–996. spectral edge, Biophys. J. 87(1), 631–639. Helmchen, F., et al. (2001) A miniature Lakowicz, J.R., et al. (1999) Advances in head-mounted two-photon microscope high- fluorescence spectroscopy: multi-photon resolution brain imaging in freely moving excitation, engineered proteins, modulation animals, Neuron 31(6), 903–912. sensing and microsecond rhenium metal- Helmchen, F., Tank, D.W., Denk, W. (2002) ligand complexes, Acta. Phys. Pol. A 95(1), Enhanced two-photon excitation through 179–196. optical fiber by single-mode propagation in Larson, D.R., et al. (2003) Water-soluble alargecore,Appl. Opt. 41(15), 2930–2934. quantum dots for multiphoton fluorescence Hentschel, M., et al. (2001) Attosecond metrol- imaging in vivo, Science 300(5624), 1434–1436. ogy, Nature 414(6863), 509–513. Lechleiter, J.D., Lin, D.T., Sieneart, I. (2002) Hockberger, P.E., et al. (1999) Activation Multi-photon laser scanning microscopy using of flavin-containing oxidases underlies an acoustic optical deflector, Biophys. J. 83(4), light-induced production of H2O2 in 2292–2299. 86 Two-photon Microscopy and Imaging

Lipp, P., Niggli, E. (1998) Fundamental calcium Muller, M., et al. (1998) Dispersion pre- release events revealed by two-photon compensation of 15 femtosecond optical excitation photolysis of caged calcium in pulses for high-numerical- objectives, guinea-pig cardiac myocytes, J. Physiol. J. Microsc. (Oxford) 191, 141–150. (London) 508(3), 801–809. Neil, M.A.A., et al. (2000) Adaptive aberration Lippitz, M., et al. (2002) Two-photon excitation correction in a two-photon microscope, J. microscopy of tryptophan-containing proteins, Microsc. (Oxford) 200, 105–108. Proc. Natl. Acad. Sci. U.S.A. 99(5), 2772–2777. Nielsen, T., et al. 2001 High efficiency Lombardo, J.A., et al. (2003) Amyloid-beta anti- beam splitter for multifocal multiphoton body treatment leads to rapid normalization microscopy, J. Microsc. 201, 368–376. of plaque-induced neuritic alterations, J. Neu- Nitsch, R., et al. (2004) Direct impact of T cells on rosci. 23(34), 10879–10883. neurons revealed by two-photon microscopy Maiti, S., et al. (1997) Measuring serotonin in living brain tissue, J. Neurosci. 24(10), distribution in live cells with three-photon 2458–2464. excitation, Science 275(5299), 530–532. Norris, T.B. (1992) Femtosecond pulse amplifica- Maletic-Savatic, M., Malinow, R., Svoboda, K. tion at 250 kHz with a Ti-sapphire regenerative (1999) Rapid dendritic morphogenesis in CA1 amplifier and application to continuum gen- hippocampal dendrites induced by synaptic eration, Opt. Lett. 17(14), 1009–1011. activity, Science 283(5409), 1923–1927. Ouzounov, D.G., et al. (2002) Delivery of Margrie, T.W., et al. (2003) Targeted whole-cell nanojoule femtosecond pulses through large- recordings in the mammalian brain in vivo, core microstructured fibers, Opt. Lett. 27(17), Neuron 39(6), 911–918. 1513–1515. Masters, B.R., Kriete, A., Kukulies, J. (1993) Pastirk, I., et al. (2003) Selective two-photon Ultraviolet confocalfluoresence microscopy in microscopy with shaped femtosecond pulses, the in vitro cornea: redox metabolic imaging, Opt. Express 11(14), 1695–1701. Appl. Opt. 32(4), 592–596. Patterson, G.H., Piston, D.W. (2000) Photo- Masters, B.R., So, P.T.C., Gratton, E. (1997) Mul- bleaching in two-photon excitation mi- tiphoton excitation fluorescence microscopy croscopy, Biophys. J. 78(4), 2159–2162. and spectroscopy of in vivo human skin, Bio- Pawley, J., (Ed.) 1995 Handbook of Biological phys. J. 72(6), 2405–2412. Confocal Microscopy, 2nd edition, Kluwer Matsuzaki, M., et al. (2001) Dendritic spine geometry is critical for AMPA receptor Academic Publishers, Norwell, MA. expression in hippocampal CA1 pyramidal Pelliccioli, A.P., Wirz, J 2002 Photoremovable neurons, Nat. Neurosci. 4(11), 1086–1092. protecting groups: reaction mechanisms and McClain, W.M. Excited state symmetry applications, Photochem. Photobiol. Sci. 1(7), assignment through polarized two-photon 441–458. absorption studies of fluids, J. Chem. Phys. Piston, D.W., et al. (1994) 2-photon-excitation 1971. 55(6), 2789–2796. fluorescence imaging of 3-dimensional Mempel, T.R., et al. (2004) In vivo imaging of calcium-ion activity, Appl. Opt. 33(4), 662–669. leukocyte trafficking in blood vessels and Piston, D.W., Knobel, S.M. (1999) Real-time tissues, Curr. Opin. Immunol. 16(4), 406–417. analysis of glucose metabolism by microscopy, Miller, M.J., et al. (2002) Two-photon imaging Trends Endocrinol. Metab. 10(10), 413–417. of lymphocyte motility and antigen response Piston, D.W., Masters, B.R., Webb, W.W. (1995) in intact lymph node, Science 296(5574), 3-dimensionally resolved Nad(P)H cellular 1869–1873. metabolic redox imaging of the in-situ Minsky, M. (1961) Microscopy Apparatus, in US cornea with 2-photon excitation laser-scanning Patent, 3013467. microscopy, J. Microsc. (Oxford) 178, 20–27. Minta, A., Kao, J.P., Tsien, R.Y. (1989) Fluores- Potter, S.M., Pine, J., Fraser, S.E. (1996) Neural cent indicators for cytosolic calcium based on transplant staining with DiI and vital imaging rhodamine and fluorescein chromophores, J. by 2-photon laser-scanning microscopy, Biol. Chem. 264(14), 8171–8178. Scanning Microsc. Suppl. 10, 189–199. Miyawaki, A., et al. (1997) Fluorescent indicators Prasher, D.C., et al. (1992) Primary structure for Ca2+ based on green fluorescent proteins of the Aequorea victoria green-fluorescent and calmodulin, Nature 388(6645), 882–887. protein, Gene 111(2), 229–233. Two-photon Microscopy and Imaging 87

Shen, Y.R. (1984) The Principles of Nonlinear Theer, P., Hasan, M.T., Denk, W. (2003) Two- Optics, Wiley, New York. photon imaging to a depth of 1000 mu m in Sheppard, C.J.R. (1980) Scanning optical living brains by use of a Ti:Al2O3 regenerative microscope, Electron. Power 26(2), 166–172. amplifier, Opt. Lett. 28(12), 1022–1024. Shirakawa, A., Sakane, I., Kobayashi, T. (1998) Trachtenberg, J.T., et al. (2002) Long-term in vivo Pulse-front-matched optical parametric ampli- imaging of experience-dependent synaptic fication for sub-10-fs pulse generation tunable plasticity in adult cortex, Nature 420(6917), in the visible and near infrared, Opt. Lett. 788–794. 23(16), 1292–1294. Treacy, E. (1969) Optical Pulse Compression Spence, D.E., Kean, P.N., Sibbett, W. (1991) 60- With Diffraction Gratings, IEEE J. Quantum fsec pulse generation from a self-mode-locked Electron. QE-5(9), 454–458. Ti:sapphire laser, Opt. Lett. 16(1), 42–44. Tsien, R.Y. (1981) A non-disruptive technique Squier, J., Muller, M. (2001) High resolution for loading calcium buffers and indicators into nonlinear microscopy: a review of sources and cells, Nature 290(5806), 527–528. methods for achieving optimal imaging, Rev. Tsien, R.Y. (1989) Fluorescent-Probes of Cell Sci. Instrum. 72(7), 2855–2867. Signaling, Annu. Rev. Neurosci. 12, 227–253. Squirrell, J.M., et al. (1999) Long-term two- White, J., Amos, W., Fordham, M. (1987) An photon fluorescence imaging of mammalian evaluation of confocal versus conventional embryos without compromising viability, Nat. imaging of biological structures by Biotechnol. 17(8), 763–767. fluorescence light microscopy, J. Cell Biol. 105, Stelzer, E.H.K., et al. (1994) Nonlinear absorp- 41–48. tion extends confocal fluorescence microscopy Wilson, T., Sheppard, C. (1984) Theory and into the ultra-violet regime and confines the Practice of Scanning Optical Microscopy, illumination volume, Opt. Commun. 104(4–6), Academic Press, London. 223–228. Xu, C., Webb, W.W. (1996) Measurement of two- Stosiek, C., et al. (2003) In vivo two-photon photon excitation cross sections of molecular calcium imaging of neuronal networks, Proc. fluorophores with data from 690 to 1050 nm, Natl. Acad. Sci. U.S.A. 100(12), 7319–7324. J. Opt. Soc. Am. B 13(3), 481–491. Svaasand, L.O., Ellingsen, R. (1983) Optical Yaroslavsky, A.N., et al. (2002) Optical properties properties of human brain, Photochem. of selected native and coagulated human Photobiol. 38(3), 293–299. brain tissues in vitro in the visible and near Svoboda, K., et al. (1997) In vivo dendritic infrared spectral range, Phys. Med. Biol. 47(12), calcium dynamics in neocortical pyramidal 2059–2073. neurons, Nature 385(6612), 161–165. Yasuda, R., et al. (2004) Imaging calcium Svoboda, K., Tank, D.W., Denk, W. (1996) concentration dynamics in small neuronal Direct measurement of coupling between compartments, Sci. STKE 2004(219), pl5. dendritic spines and shafts, Science 272(5262), Yuste, R., Denk, W. (1995) Dendritic spines as 716–719. basic functional units of neuronal integration, Szmacinski, H., Gryczynski, I., Lakowicz, J.R. Nature 375(6533), 682–684. (1998) Spatially localized ballistic two-photon Zhang, J., et al. (2002) Creating new fluorescent excitation in scattering media, Biospectroscopy probes for , Nat. Rev. Mol. Cell Biol. 4(5), 303–310. 3(12), 906–918. Tan, Y.P., et al. (1999) Fast scanning and Zumbusch, A., Holtom, G.R., Xie, X.S. (1999) efficient photodetection in a simple two- Three-dimensional vibrational imaging by photon microscope, J. Neurosci. Methods coherent anti-stokes Raman scattering, Phys. 92(1–2),123–135. Rev. Lett. 82(20), 4142–4145.

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