Microscopic Imaging

Nephron Exp Nephrol 2006;103:e33–e40 Published online: March 10, 2006 DOI: 10.1159/000090614

Principles of Multiphoton Microscopy

Kenneth W. Dunn Pamela A. Young

Department of Medicine, Division of Nephrology, Indiana University School of Medicine, Indianapolis, Ind., USA

Key Words Introduction Multiphoton microscopy Fluorescence In vivo imaging Three-dimensional imaging Intravital Fluorescence microscopy has become a vital compo- microscopy nent of biomedical research, allowing investigators to lo- calize specifi c molecules and characterize cell physiology at subcellular resolution. The extended reach and low Abstract photon toxicity of multiphoton microscopy now offers Multiphoton fl uorescence microscopy is a powerful, im- biomedical researchers the capability of characterizing portant tool in biomedical research that offers low pho- cellular and subcellular processes deep into tissues in ton toxicity and higher spatial and temporal resolution three dimensions and even in the context of tissues and than other in vivo imaging modalities. The capability to organs in living animals. Here we present a basic intro- collect images hundreds of micrometers into biological duction to the principles, biological applications, practi- tissues provides an invaluable tool for studying cellular cal aspects and future of multiphoton fl uorescence mi- and subcellular processes in the context of tissues and croscopy in biomedical research. organs in living animals. Multiphoton microscopy is based upon two-photon excitation of fl uorescence that occurs only in a sub-femtoliter volume at the focus; by Principles and Development of Multiphoton scanning the focus through a sample, 2- and 3-dimen- Fluorescence Microscopy sional images can be collected. The complex 3-dimen- sional organization of the kidney makes it especially ap- Conventional fl uorescence is stimulated by the absorp- propriate for multiphoton microscopic analysis, which tion of a photon by a fl uorophore, raising an electron to an has been used to characterize numerous aspects of renal excited energy state which, when it returns to ground state, physiology and pathophysiology in living rats and mice. results in the release of a photon. In contrast, multiphoton However, the ability to collect fl uorescence images deep fl uorescence microscopy is based upon the simultaneous into biological tissues raises unique problems not en- absorption of two, low-energy photons by a molecule countered in other forms of optical microscopy, includ- (fi g. 1 ). While the energy of either of these low-energy pho- ing issues of probe access, and tissue optics. Future im- tons is insuffi cient to excite an electron, their combined provements in multiphoton fl uorescence microscopy energy is enough to raise an electron to the excited state will involve optimizing objectives for the unique charac- and thus stimulate fl uorescence. This process was theo- teristics of multiphoton fl uorescence imaging, improv- retically predicted in the PhD dissertation of Maria Goep- ing the speed at which images may be collected and pert-Mayer in 1931, part of the body of work that resulted extending the depth to which imaging may be con- in her receiving the fi rst Nobel Prize in theoretical physics ducted. awarded to a woman [Goeppert-Mayer, 1931]. Copyright © 2006 S. Karger AG, Basel

© 2006 S. Karger AG, Basel Kenneth W. Dunn, PhD 1660–2129/06/1032–0033$23.50/0 Nephrology Division, Indiana University School of Medicine Fax +41 61 306 12 34 950 W. Walnut St., R2-202B, Indianapolis, IN 46202 (USA) E-Mail [email protected] Accessible online at: Tel. +1 317 278 0436, Fax +1 317 274 8575 www.karger.com www.karger.com/nee E-Mail [email protected] One-photon Two-photon

S1 S1

Excitation Fluorescence Excitation Fluorescence photon emission photons emission photon photon

Fig. 1. Jablonski diagram demonstrating one- and two-photon fl uorescence excita- tion. Two-photon excitation results from S S the simultaneous absorption of two low- 0 0 energy photons by a fl uorophore.

Fluorophore Excited fluorophore One-photon Two-photon

Fig. 2. Fluorescence excitation for one- and two-photon microscopy. In confocal mi- croscopy, a continuous wave ultraviolet or visible light laser excites fl uorophores throughout the volume. In two-photon mi- croscopy, an infrared laser provides pulsed illumination such that the density of pho- tons suffi cient for simultaneous absorption of two photons by fl uorophores only occurs at the focal point.

In order to stimulate an electronic transition, the two it was not until suffi ciently powerful lasers were devel- photons must arrive at the fl uorophore within approxi- oped 30 years later that Dr. Goeppert-Mayer’s predic- mately an attosecond (10 –18 s) of one another [McCain, tions of multiphoton fl uorescence excitation were fi nally 1970], which under most circumstances is a very unlike- experimentally validated [Kaiser and Garret, 1961]. ly event. Winfried Denk, one of the inventors of multi- However, the high power that made these early lasers photon microscopy, presented an illustrative numerical capable of stimulating multiphoton fl uorescence excita- example that demonstrates this point [Denk and Svobo- tion also made them incompatible with biological mate- da, 1997]. A molecule of rhodamine, exposed to direct rial. Consequently, the utility of multiphoton fl uores- sunlight, will absorb a single photon around once per sec- cence to biological microscopy was not realized for yet ond but will experience a two-photon absorption around another 30 years, with the development of ultra-short once every 10 million years. Thus, to generate detectable pulsed-laser systems [Denk et al., 1990]. These laser sys- amounts of fl uorescence, multiphoton fl uorescence exci- tems emit light in brief, intense pulses (typically between tation requires an enormous photon fl ux. Consequently, 100 and 2,000 fs in length), at a frequency of around

e34 Nephron Exp Nephrol 2006;103:e33–e40 Dunn/Young

Table 1. Comparison of confocal and multiphoton microscopy

Confocal microscopy Multiphoton microscopy

Excitation source Ultraviolet or visible laser Pulsed infrared laser Limited number of wavelengths Continuously variable wavelength Low cost High cost Effective imaging depth Typically <20 m Up to 500–600 ma Spatial resolution Theoretically up to Theoretically up to (full width at half-maximum) 0.14 m laterally 0.23 m laterally 0.57 m axiallyb 0.93 m axiallyb Sensitivity to scattering High Low Photobleaching and phototoxicity High – occurs throughout tissue volume Low – restricted to focal plane Image capture rate Typically low, higher speeds possible via array Low, array scanning limited to thin scanning samples

a See Kleinfeld et al. [1998] and Helmchen et al. [1999]. b Assuming 488 nm excitation for confocal and 900 nm excitation for multiphoton [Jonkman and Stelzer, 2002]. Note that the reso- lution of confocal and multiphoton systems are more similar in practice [e.g. Centonze and White, 1998].

80 MHz (fi g. 2). In doing this, pulsed lasers provide the microscopy. Since the size of a diffraction limited spot is high peak photon fl ux necessary to stimulate detectable proportional to the wavelength of light, one might expect fl uorescence, but because their duty cycle is so small the resolution of multiphoton microscopy to be approxi- (typically approximately 0.001%), the average power is low mately 2-fold worse than that of confocal microscopy. In enough to avoid damage to tissues or fl uorescent probes. fact, due to the two-photon mechanism, the calculation In a multiphoton microscope system the probability of is somewhat more complicated, but still predicts a resolu- multiphoton absorption is further increased by focusing tion that is approximately 60% worse than that of confo- the beam down to a diffraction-limited spot through a cal microscopy ( table 1). However, resolution is infl u- high numerical aperture microscope objective. Because enced by a number of additional factors, including image the photon fl ux decreases with the fourth power of dis- noise and background, so that in actual practice, the res- tance from the focus, multiphoton fl uorescence excitation olutions of the two systems are similar [Centonze and is effectively limited to a sub-femtoliter volume at the White, 1998; Konig, 2000; Zipfel et al., 2003b]. focus. Thus, one can collect optical sections from a sam- Multiphoton microscopy has several advantages over ple volume by raster-scanning the focus across the sam- confocal microscopy for collecting images deep within ple, and by translating the focus through the sample, one biological samples. First, since fl uorescence excitation is can collect 3-dimensional image volumes. limited to a single spot in the sample volume, no confocal The combined effects of pulsing and focusing the laser aperture is needed to reject out-of-plane fl uorescence. are such that under typical use, the average peak photon This is a signifi cant advantage for imaging thick tissues fl ux is more than a million times that at the surface of the where light scattering can redirect emissions away from sun. While this sounds ridiculous, the photon pulse is so the confocal aperture, reducing sensitivity and contrast. brief, and its volume is so small, that estimates indicate Since excitation is localized to a single point, fl uorescence that, even with 100 times this power, illuminating the emissions can be collected using one or more detectors focus for one full second would increase the temperature located close to the microscope objective to optimize col- of water by only 0.2 K [Schonle and Hell, 1998]. Indeed, lection of scattered light. In addition to more effi cient multiphoton microscopy has been found to be remark- collection of fl uorescence, multiphoton microscopy also ably non-toxic. Squirrell et al. [1999] collected multipho- offers more effi cient stimulation of fl uorescence deep into ton fl uorescence images of embryonic hamsters every biological tissues. As compared with the visible light used 15 min over a period of 24 h without affecting their de- in confocal microscopy, the near-infrared light used to velopment. stimulate multiphoton fl uorescence is both scattered and Multiphoton microscopy thus provides a method of absorbed less by biological tissues, so that fl uorescence optical sectioning that compares favorably with confocal can be excited deeper into biological samples [Konig,

Multiphoton Microscopy Nephron Exp Nephrol 2006;103:e33–e40 e35 2000]. Finally, since fl uorescence is stimulated only at the multiphoton microscopic analysis. Phillips et al. [2001, focal point, photobleaching is likewise limited to this 2004] have used multiphoton microscopy to characterize point. The lack of out-of-plane photobleaching makes renal development in a mouse model of polycystic kidney multiphoton microscopy particularly amenable to collec- disease. Because the kidney can be easily immobilized tion of 3-dimensional image volumes, which when col- and apposed to a coverglass on the stage of a microscope, lected via confocal microscopy, suffer from the accumu- multiphoton microscopy can also be used to image renal lation of photobleaching of the entire volume that occurs physiology in living rats and mice [Dunn et al., 2002] and with the collection of each image plane. has been applied to studies of microvascular leakage in a Thus, for several reasons, multiphoton fl uorescence rat model of renal ischemia [Sutton et al., 2003, 2005], microscopy is superior to confocal microscopy for imag- folic acid uptake and transport [Sandoval et al., 2004] and ing deep into biological tissues (a detailed demonstration organic anion transport in rat proximal tubule cells [Tan- can be found in Centonze and White [1998]). Examples ner et al., 2004]. Mik et al. [2004] have used multiphoton of how biomedical researchers have utilized multiphoton excitation to characterize local oxygen concentrations in microscopy for imaging deep into biological tissues are kidney cortex of living rats. The laboratory of Peti-Pe- described in the next section. terdi has used multiphoton microscopy to characterize the function of isolated living glomeruli, arterioles, and cortical and medullary tissues [Peti-Peterdi et al., 2004; Utilization of Multiphoton Fluorescence Peti-Peterdi, 2005]. Microscopy in Biomedical Research

The deployment of multiphoton microscopy in bio- Practical Multiphoton Microscopy medical research has continued to be driven by technical developments in lasers. The original multiphoton micro- On fi rst principles, one might expect that multiphoton scope system utilized a fussy dye laser system that was fl uorescence microscopy differs from conventional fl uo- too technically challenging for most biomedical research- rescence microscopy only in the use of near-infrared il- ers [Denk et al., 1990]. Thus, multiphoton microscopy lumination to excite fl uorescence. Indeed, many current was limited to very few laboratories until the develop- multiphoton microscope systems are simply confocal mi- ment of solid-state titanium-sapphire lasers in 1992 croscopes modifi ed for infrared illumination. However, [Curley et al., 1992]. Still somewhat challenging to multiphoton microscopy is unique in several respects. In the average biologist, these systems became progressive- addition, the ability to collect fl uorescence images deep ly easier to operate in the past 10 years, culminating in into biological tissues raises unique problems not encoun- the recent development of ‘closed-box’ computer-con- tered in other forms of optical microscopy. trolled systems that require almost no attention from The fi rst challenge concerns fl uorescent probes for the user. multiphoton microscopy. The process of two-photon ex- With the development of commercial systems in 1996, citation is generally introduced with the example that the multiphoton microscopy fi nally evolved from a method fl uorescence ordinarily excited by a single photon of a limited to laboratories specialized in advanced biopho- particular energy can also be excited by two photons, each tonics to one accessible to a broad range of biomedical with half that energy. Thus one would expect that effi cient researchers. Since that time, biomedical researchers have multiphoton excitation could be accomplished by simply applied multiphoton microscopy to a variety of studies, doubling the wavelength used for one-photon excitation. including some particularly exciting analyses of cellular However, the ‘two-photon action cross section’ (the prop- and intracellular processes in living animals. Multiphoton erty used to describe the probability of two-photon ab- microscopy has been used for in vivo analysis of dendrit- sorption) can differ dramatically from this simple rela- ic spine development, cortical blood fl ow, senile plaque tionship. So, for example, while the two-photon excita- clearance and dendritic calcium dynamics in the brain, tion profi les for probes such as lucifer yellow and Fura-2 blood fl ow, angiogenesis and vascular permeability in tu- closely approximate two times their single photon excita- mors and T-cell traffi cking in lymph nodes (summarized tions, probes such as fl uorescein and rhodamine show in Denk and Svoboda [1997] and Zipfel et al. [2003b]). two-photon excitation optima that are not obvious from The complex 3-dimensional organization of organs their corresponding one-photon excitation spectra. Thus such as the kidney makes it especially appropriate for before designing a multiphoton fl uorescence microscopy

e36 Nephron Exp Nephrol 2006;103:e33–e40 Dunn/Young

experiment, one should consult the analyses published by collected, unlike a confocal microscope where the out-of- the Cornell research group [Xu et al., 1996; Zipfel et al., plane fl uorescence must be fi ltered out with the confocal 2003a, 2003b] to identify the appropriate excitation pinhole. Consequently, multiphoton fl uorescence emis- wavelength. sions can and should be collected with detectors located Even after one has selected a fl uorescent probe, the as close to the microscope objective as possible. In addi- next challenge may be to ensure that the probe has access tion to avoiding the inevitable transmission losses of the to its targets in the sample. The ability to image deep into various fi lters and refl ectors in the de-scanning mecha- biological specimens raises unique issues of preservation nism of the confocal microscope, this design also more and permeability of thick, fi xed specimens. Careful ex- effectively collects scattered fl uorescence emissions that periments must be conducted to demonstrate that the are unlikely to successfully navigate the long optical path distribution of the probe in the sample is not affected by from the microscope objective to the photomultiplier de- the access of the probe to different parts of the sample. tector in the confocal scanhead. A convincing demonstra- The problem of probe access is more complicated for tion of the benefi t of using external, ‘non-descanned’ de- studies of living tissues, especially within living animals. tectors is shown in Centonze and White [1998]. In this case, probe access depends upon physiological Multiphoton excitation can be accomplished with in- methods of probe delivery. So, for example, we have frared lasers that provide pulsewidths in the range of found that intravenous injection of a membrane-poten- 100–200 fs or in the range of picoseconds. However, pi- tial-sensitive probe, rhodamine R6, brightly labels the cosecond lasers stimulate less multiphoton fl uorescence mitochondria of endothelia, but fails to label renal epi- than femtosecond lasers for the same average power. thelial cells [Dunn et al., 2002]. Since multiphoton fl uorescence excitation is proportion- One solution to the problem of labeling specifi c cells al to the square of the illumination power divided by in living tissues is to express an exogenous fl uorescent duration of the pulse, a laser producing 1-ps pulses re- protein label. This can be accomplished by generating a quires 3.16 times more power to produce the same transgenic animal expressing a fl uorescent protein under amount of fl uorescence as a laser producing 100-fs puls- the control of a tissue-specifi c promoter. For example, es. Because of patent issues, the choice of laser system is Sutton et al. [2003] examined injury to the renal micro- tied to the choice of commercial multiphoton micro- vasculature in a mouse expressing GFP under the control scope system, since ‘sub-picosecond’ multiphoton fl uo- of an endothelial specifi c promoter. Alternatively, the rescence microscopy is licensed to the Zeiss, whereas gene may be introduced locally into a group of cells. For picosecond multiphoton fl uorescence microscopy is li- example, Tanner et al. [2005] have used micropuncture- censed to Leica. The patent situation surrounding mul- mediated delivery of adenovirus to express various GFP tiphoton microscopy has unfortunately limited its tech- protein chimeras in rat kidney cells. nical development since its fi rst commercial introduc- Alternatively, one can circumvent the problem of de- tion by BioRad in 1996. livering fl uorescent reporters by using endogenous fl uo- Although their lower effi ciency would seem to con- rophores. Many endogenous proteins, such as indoleam- demn picosecond systems, in many cases multiphoton ines, fl avins, NAD(P)H and serotonin have fl uorescence microscopy is not limited by laser power. While the lasers that can be excited by multiphoton excitation [Zipfel et of most systems are capable of delivering hundreds of al., 2003a]. The environmental sensitivity of NAD(P)H milliwatts of power at the sample, powers above approx- and fl avin fl uorescence has been used to characterize cel- imately 10 mW induce cell damage, as evaluated by a lular redox in vivo in the rabbit cornea [Piston et al., variety of criteria [Konig et al., 1997; Konig, 2000; Hopt 1995] and in human skin [Masters et al., 1997]. and Neher, 2001] and powers in the range of 30–50 mW In order to realize the benefi ts of multiphoton micros- have been used to drill holes and sever intracellular struc- copy for deep tissue imaging, special attention must be tures [Konig, 2000]. As described previously, multipho- paid to the fl uorescence detection system. While confocal ton microscopy conducted at lower levels of illumination microscope systems may be modifi ed into multiphoton can be remarkably non-toxic [Squirrell et al., 1999], but microscope systems, the collection system of a confocal the damaging effects on cell viability appear to be non- microscope is poorly suited to collecting fl uorescence linear, so that damage is roughly proportional to fl uores- from deep inside biological tissues. Since multiphoton cence up to 10–15 mW, but then scaling at a higher rate fl uorescence excitation is inherently localized to a single above those levels [Koester et al., 1999; Hopt and Neher, spot in the sample, all of the emitted fl uorescence can be 2001]. High power also appears to be disproportionately

Multiphoton Microscopy Nephron Exp Nephrol 2006;103:e33–e40 e37 harmful to fl uorescent probes; photobleaching rates in- used for conventional fl uorescence and confocal micros- crease disproportionately faster than fl uorescence signals copy. over this range of powers [Patterson and Piston, 2000]. It Multiphoton microscopy places unique demands on is noteworthy that below these threshold values, photo- microscope objectives. New designs optimized for trans- toxicity is apparently mediated by a two-photon excita- mission of near-infrared light improve the effi ciency of tion process [Konig, 2000], meaning that while picosec- multiphoton fl uorescence excitation. However, opportu- ond systems excite less fl uorescence for a given average nities for further improvements in multiphoton fl uores- power, they induce proportionally less photodamage as cence performance may result from designs optimized for well. Thus, picosecond and femtosecond are effectively the fundamentally different role that the microscope equivalent under conditions where fl uorescence signals objective in multiphoton microscopy plays. Objective are limited by phototoxicity. lenses designed for conventional fl uorescence and confo- While these arguments suggest that laser power is cal microscopy are designed to focus visible wavelengths available in abundance in multiphoton microscope sys- of light down to a diffraction limited spot in the sample tems, they ignore the fact that the photon fl ux delivered plane. In contrast, objectives optimized for multiphoton to the focal point deep in a biological sample may be sig- fl uorescence should be designed to focus a brief pulse of nifi cantly reduced by scattering. Thus, laser power may a range of infrared wavelengths of light to a point in the be limiting for the deep-tissue imaging applications most sample and to do so without broadening the pulse. Im- appropriate for multiphoton microscopy. In many cases, provements are needed to decrease dispersion in objec- we have found that illumination levels must be increased tive lenses that broadens the laser pulse, decreasing the in order to collect adequate images deep in tissues. Konig effi ciency of multiphoton fl uorescence excitation. Objec- [2000] reports that laser power must be increased 10-fold tives designed for conventional fl uorescence and confocal to acquire images 100 m into a tumor equivalent to microscopy are designed to focus fl uorescence emissions those collected at the surface. Under these conditions, the down to a diffraction limited spot in the image plane. In additional effi ciency of femtosecond laser systems may contrast, objectives used for multiphoton microscopy be required. need not be designed to image fl uorescence, but rather to Other authors argue that the main factor limiting the effi ciently collect fl uorescence, both scattered and unscat- collection of images deep into tissues is scattering or ab- tered. Oheim et al. [2001] fi nd that increasing the angle sorption of fl uorescence emissions [Zipfel et al., 2003b]. of acceptance of the collection optics improves the effi - To the degree that suffi cient fl uorescence is stimulated at ciency of fl uorescence collection at depth up to 30-fold. depth, but the fl uorescence emissions are scattered and Of particular note is the observation that a low-magnifi - thus undetected, increasing laser power will have a lim- cation, high numerical aperture objective provides a 10- ited capacity for improving the signal. In fact, if adequate fold improvement in the collection of emissions deep into fl uorescence is being generated but not detected, increas- brain tissues, as compared with 60–63 ! objectives with ing laser power will probably rapidly incur fl uorescence a similar numerical aperture, but smaller acceptance an- saturation. Increasing laser power under these conditions gles. will not only fail to improve signal, but can also decrease Another factor that limits the depth at which imaging the resolution of the image as the size of the excited vol- can be conducted by multiphoton fl uorescence micros- ume is effectively increased [Zipfel et al., 2003b]. copy is the degradation of the shape of the illuminating spot, resulting from inhomogeneities in the sample. Re- cent results indicate that this problem can be ameliorated Future Developments in Multiphoton with adaptive optics. Widely used in astronomy, adaptive Microscopy optics involves the specifi c shaping of lenses to compen- sate for specimen-induced aberrations. Optimized shap- Although invented more than 15 years ago, multipho- ing of deformable lenses in the excitation light path of ton microscopy is still an immature technology, particu- multiphoton fl uorescence microscope systems has been larly as it exists in commercially available instruments. found to improve axial resolution 2-fold at a depth of While some experimental systems have been specifi cally 50 m [Marsh et al., 2003] and to extend the useful depth designed for the unique capabilities and requirements of from 150 to 800 m [Sherman et al., 2002] into an aber- multiphoton microscopy, commercial instruments still rating medium. largely utilize designs only slightly modifi ed from those

e38 Nephron Exp Nephrol 2006;103:e33–e40 Dunn/Young

Technical developments may also increase the speed priate for multiphoton microscopy, deep tissue imaging. of image acquisition in multiphoton microscopy. Com- High-speed point scanning systems depend upon high mercial systems collect reasonable sized images at a rate levels of signal that may be diffi cult to achieve deep in of 1–4 frames per second, a rate too slow to observe rap- tissues. It is unlikely that illumination levels can be in- id dynamic processes in living tissues. For some applica- creased to compensate for the low signal levels of these tions, these slow capture rates can be compensated by systems. A 30-fold increase in image capture rate will re- collecting line scans, in which a single line in the sample quire more than a 5-fold increase in power that is likely is repeatedly scanned at millisecond frequencies, an ap- to incur fl uorescence saturation, and may not be available proach that has been used to characterize calcium tran- from the laser source. Multifocal multiphoton micro- sients [Helmchen et al., 1999] and blood fl ow [Kleinfeld scope systems may likewise be limited by power when et al., 1998; Brown et al., 2001] in living rats. While im- imaging deep into tissues, and scattered emissions will proving speed, this technique provides limited spatial in- compromise both sensitivity and image contrast. formation. Alternative designs of multiphoton microscopes, with faster scanning systems, are capable of signifi cantly in- Deployment of Multiphoton Microscopy creasing the rate of image capture, supporting image cap- ture at 15–100 frames per second [Fan et al., 1999; Kim A survey of the published literature conducted in 2003 et al., 1999; Nguyen et al., 2001]. However, the utility of indicates that while the use of multiphoton microscopy these systems is limited to samples in which signal is not has grown exponentially over the past 15 years, most pub- limiting, since as the speed of collection increases, the lications come from a relatively few laboratories [Zipfel period of light collection proportionately decreases. et al., 2003b]. To some extent this slow deployment of An alternative approach to high-speed scanning mi- multiphoton microscopy may refl ect the cost and/or tech- croscopy is to scan multiple foci simultaneously across nical complexity of multiphoton microscope systems. the sample. In this way speed can be increased without However, the biomedical application of multiphoton mi- necessarily sacrifi cing signal. One problem with this ap- croscopy is unusual in that it involves many different proach is that as the number and density of beams are kinds of expertise. Our experience indicates that fully re- increased, they begin to overlap with each other, resulting alizing the unique potential of multiphoton microscopy in excitation of fl uorescence outside the focal plane, and requires an unusual degree of collaboration, not only be- corresponding decreases in axial resolution and image tween microscopists and cell biologists, but also with cli- contrast. An elegant solution to this problem is to tem- nicians and individuals with skills in animal handling, porally offset the excitation of adjacent foci by 250– tissue histology and digital image processing. Thus it may 1,000 fs, thus preventing reinforcement of excitation be- be diffi cult to achieve the critical mass of expertise neces- tween them [Andressen et al., 2001; Nielsen et al., 2001; sary to fully exploit multiphoton microscopy. However, Egner et al., 2002]. these diffi culties are more than compensated by the enor- The major drawback of this approach is that it is poor- mous rewards of this technique, providing the ability to ly suited to thick, scattering samples. In order to realize visualize and quantify cellular and intracellular processes the speed benefi t, fl uorescence is collected via an imaging in three dimensions and in living animals. detector, such as a CCD. Whereas unscattered fl uores- cence will be effi ciently focused to the appropriate detec- tor element, and thus sharply imaged, scattered fl uores- cence will be directed to incorrect detector elements, re- ducing both resolution and image contrast. In addition, dividing the illumination between multiple beams de- pends upon a surplus of power. As discussed above, this is likely to be reasonable for thin samples, but perhaps not for thick samples where illumination levels must be increased. Thus, while there are solutions to the problem of slow image acquisition in multiphoton microscopy, they do not seem to be appropriate to the conditions most appro-

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