Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience
2006; Cell Press; Volume: 50; Issue: 6 Linguagem: Inglês
10.1016/j.neuron.2006.05.019
ISSN1097-4199
Autores Tópico(s)Neuroscience and Neuropharmacology Research
ResumoThe brain is complex and dynamic. The spatial scales of interest to the neurobiologist range from individual synapses (∼1 μm) to neural circuits (centimeters); the timescales range from the flickering of channels (less than a millisecond) to long-term memory (years). Remarkably, fluorescence microscopy has the potential to revolutionize research on all of these spatial and temporal scales. Two-photon excitation (2PE) laser scanning microscopy allows high-resolution and high-sensitivity fluorescence microscopy in intact neural tissue, which is hostile to traditional forms of microscopy. Over the last 10 years, applications of 2PE, including microscopy and photostimulation, have contributed to our understanding of a broad array of neurobiological phenomena, including the dynamics of single channels in individual synapses and the functional organization of cortical maps. Here we review the principles of 2PE microscopy, highlight recent applications, discuss its limitations, and point to areas for future research and development. The brain is complex and dynamic. The spatial scales of interest to the neurobiologist range from individual synapses (∼1 μm) to neural circuits (centimeters); the timescales range from the flickering of channels (less than a millisecond) to long-term memory (years). Remarkably, fluorescence microscopy has the potential to revolutionize research on all of these spatial and temporal scales. Two-photon excitation (2PE) laser scanning microscopy allows high-resolution and high-sensitivity fluorescence microscopy in intact neural tissue, which is hostile to traditional forms of microscopy. Over the last 10 years, applications of 2PE, including microscopy and photostimulation, have contributed to our understanding of a broad array of neurobiological phenomena, including the dynamics of single channels in individual synapses and the functional organization of cortical maps. Here we review the principles of 2PE microscopy, highlight recent applications, discuss its limitations, and point to areas for future research and development. Fluorescence microscopy occupies a unique niche in biological microscopy. Fluorescent objects can be selectively excited and visualized, even in living systems (Lichtman and Conchello, 2005Lichtman J.W. Conchello J.A. Fluorescence microscopy.Nat. Methods. 2005; 2: 910-919Crossref PubMed Scopus (296) Google Scholar). The sensitivity of fluorescence detection is sufficiently high so that single fluorescent molecules can be detected in the presence of 1011 nonfluorescent molecules (e.g., water, amino acids, lipids) (Eigen and Rigler, 1994Eigen M. 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Dendritic spines as individual neuronal compartments for synaptic Ca2+ responses.Nature. 1991; 354: 73-76Crossref PubMed Google Scholar) and track spatially complex dynamics over centimeters of cortical tissue (Grinvald and Hildesheim, 2004Grinvald A. Hildesheim R. VSDI: a new era in functional imaging of cortical dynamics.Nat. Rev. Neurosci. 2004; 5: 874-885Crossref PubMed Scopus (230) Google Scholar). Whenever possible, neurons need to be studied in their natural habitat, and intact brain tissue is an especially challenging place for light microscopy. In wide-field fluorescence microscopy, contrast and resolution are degraded by strong scattering (Denk and Svoboda, 1997Denk W. Svoboda K. Photon upmanship: why multiphoton imaging is more than a gimmick.Neuron. 1997; 18: 351-357Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). Confocal microscopy can overcome some of the effects of scattering, since the detector pinhole rejects fluorescence from off-focus locations (Conchello and Lichtman, 2005Conchello J.A. Lichtman J.W. Optical sectioning microscopy.Nat. Methods. 2005; 2: 920-931Crossref PubMed Scopus (233) Google Scholar, Denk and Svoboda, 1997Denk W. Svoboda K. Photon upmanship: why multiphoton imaging is more than a gimmick.Neuron. 1997; 18: 351-357Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar). However, scanning a single section excites, and thereby damages, the entire specimen. Furthermore, the pinhole also rejects signal photons emanating from the focus that are scattered on their way out of the tissue. Deep in tissue, confocal microscopy becomes unacceptably wasteful in terms of signal photons (Centonze and White, 1998Centonze V.E. White J.G. Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging.Biophys. J. 1998; 75: 2015-2024Abstract Full Text Full Text PDF PubMed Google Scholar, Conchello and Lichtman, 2005Conchello J.A. Lichtman J.W. Optical sectioning microscopy.Nat. Methods. 2005; 2: 920-931Crossref PubMed Scopus (233) Google Scholar). Compensating for signal-loss with increased fluorescence excitation leads to phototoxicity and photobleaching. Wide-field and confocal microscopy are thus techniques that are best applied to thin specimens, such as cultured preparations or the most superficial cell layer in a tissue (<20 μm) (Lichtman et al., 1987Lichtman J.W. Magrassi L. Purves D. Visualization of neuromuscular junctions over periods of several months in living mice.J. Neurosci. 1987; 7: 1215-1222Crossref PubMed Google Scholar). Experiments deeper in tissue benefit from two-photon excitation (2PE; also referred to as two-photon, or multiphoton) microscopy, which allows high-resolution and high-contrast fluorescence microscopy deep in the brain (Denk et al., 1994Denk W. Delaney K.R. Gelperin A. Kleinfeld D. Strowbridge B.W. Tank D.W. Yuste R. Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.J. Neurosci. Methods. 1994; 54: 151-162Crossref PubMed Scopus (185) Google Scholar). 2PE microscopy was invented about 15 years ago (Denk et al., 1990Denk W. Strickler J.H. Webb W.W. Two-photon laser scanning microscopy.Science. 1990; 248: 73-76Crossref PubMed Google Scholar). More than one thousand publications have employed, developed, or reviewed 2PE microscopy (Denk and Svoboda, 1997Denk W. Svoboda K. Photon upmanship: why multiphoton imaging is more than a gimmick.Neuron. 1997; 18: 351-357Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, So et al., 2000So P.T. Dong C.Y. Masters B.R. Berland K.M. Two-photon excitation fluorescence microscopy.Annu. Rev. Biomed. Eng. 2000; 2: 399-429Crossref PubMed Google Scholar, Zipfel et al., 2003Zipfel W.R. Williams R.M. Webb W.W. Nonlinear magic: multiphoton microscopy in the biosciences.Nat. Biotechnol. 2003; 21: 1369-1377Crossref PubMed Scopus (1376) Google Scholar, Helmchen and Denk, 2002Helmchen F. Denk W. New developments in multiphoton microscopy.Curr. Opin. Neurobiol. 2002; 12: 593-601Crossref PubMed Scopus (111) Google Scholar, Helmchen and Denk, 2005Helmchen F. Denk W. Deep tissue two-photon microscopy.Nat. Methods. 2005; 2: 932-940Crossref PubMed Scopus (1038) Google Scholar). In this Primer, we will follow a brief introduction of the principles of 2PE with a discussion of technical advances and applications to neurobiology. Throughout we will point out the limitations of 2PE microscopy and suggest areas for future development. Photobleaching and phototoxicity, together referred to as photodamage, limit the application of fluorescence microscopy to living systems. Each excitation event carries the risk of photodamage. Optimizing fluorescence microscopy often means to minimize photodamage by maximizing the probability of detecting a signal photon per excitation event. Compared to other techniques, 2PE microscopy dramatically improves the detection of signal photons per excitation event, especially when imaging deep in highly scattering environments. In 2PE of fluorescence, two low-energy photons (typically from the same laser) cooperate to cause a higher-energy electronic transition in a fluorescent molecule (Figure 1A). 2PE is a nonlinear process in that the absorption rate depends on the second power of the light intensity. In a focused laser, the intensity is highest in the vicinity of the focus and drops off quadratically with distance above and below. As a result, fluorophores are excited almost exclusively in a tiny diffraction-limited focal volume (Figure 1B). If the beam is focused by a high numerical aperture (NA) objective, the vast majority of fluorescence excitation occurs in a focal volume that can be as small as ∼0.1 μm3 (Zipfel et al., 2003Zipfel W.R. Williams R.M. Webb W.W. Nonlinear magic: multiphoton microscopy in the biosciences.Nat. Biotechnol. 2003; 21: 1369-1377Crossref PubMed Scopus (1376) Google Scholar). The key consequence of localization of excitation is three-dimensional contrast and resolution (comparable to confocal microscopy) without the necessity for spatial filters in the detection path (e.g., the detector pinhole in the confocal microscope) (Wilson and Sheppard, 1984Wilson T. Sheppard C. Theory and Practice of Scanning Optical Microscopy. Academic Press, New York1984Google Scholar, Denk et al., 1990Denk W. Strickler J.H. Webb W.W. Two-photon laser scanning microscopy.Science. 1990; 248: 73-76Crossref PubMed Google Scholar). To generate an image, the laser is scanned over the specimen. Since the excitation occurs only in the focal volume, all fluorescence photons captured by the microscope objective constitute useful signal. When imaging in thick specimens, the signal yield per excitation event is enhanced (Figure 1B). The properties of 2PE discussed so far are independent of scattering (Denk et al., 1990Denk W. Strickler J.H. Webb W.W. Two-photon laser scanning microscopy.Science. 1990; 248: 73-76Crossref PubMed Google Scholar). When excitation photons enter tissue, their paths are altered by inhomogeneities in the index of refraction (Denk et al., 1994Denk W. Delaney K.R. Gelperin A. Kleinfeld D. Strowbridge B.W. Tank D.W. Yuste R. Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.J. Neurosci. Methods. 1994; 54: 151-162Crossref PubMed Scopus (185) Google Scholar, Denk and Svoboda, 1997Denk W. Svoboda K. Photon upmanship: why multiphoton imaging is more than a gimmick.Neuron. 1997; 18: 351-357Abstract Full Text Full Text PDF PubMed Scopus (381) Google Scholar, Helmchen and Denk, 2005Helmchen F. Denk W. Deep tissue two-photon microscopy.Nat. Methods. 2005; 2: 932-940Crossref PubMed Scopus (1038) Google Scholar). Depending on the type and age of the tissue and the wavelength of the light, about half of the incident photons are scattered every 50–200 μm (Oheim et al., 2001Oheim M. Beaurepaire E. Chaigneau E. Mertz J. Charpak S. Two-photon microscopy in brain tissue: parameters influencing the imaging depth.J. Neurosci. Methods. 2001; 111: 29-37Crossref PubMed Google Scholar, Yaroslavsky et al., 2002Yaroslavsky A.N. Schulze P.C. Yaroslavsky I.V. Schober R. Ulrich F. Schwarzmaier H.J. Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range.Phys. Med. Biol. 2002; 47: 2059-2073Crossref PubMed Scopus (179) Google Scholar, Kleinfeld et al., 1998Kleinfeld D. Mitra P.P. Helmchen F. Denk W. Fluctuations and stimulus-induced changes in blood flow observed in individual capillaries in layers 2 through 4 of rat neocortex.Proc. Natl. Acad. Sci. USA. 1998; 95: 15741-15746Crossref PubMed Scopus (356) Google Scholar). The scattering of excitation light effectively reduces the light delivered to form the diffraction-limited focus (Figure 1B). Scattering also perturbs the trajectories of fluorescence photons on their way out of the tissue (Figure 1C). Compared to one-photon techniques, 2PE provides three key advantages for microscopy in scattering specimens (Denk et al., 1994Denk W. Delaney K.R. Gelperin A. Kleinfeld D. Strowbridge B.W. Tank D.W. Yuste R. Anatomical and functional imaging of neurons using 2-photon laser scanning microscopy.J. Neurosci. Methods. 1994; 54: 151-162Crossref PubMed Scopus (185) Google Scholar). First, the excitation wavelengths used in 2PE microscopy, deep red and near IR, penetrate tissue better than the visible wavelengths used in one-photon microscopy. This improved penetration is due to reduced scattering and reduced absorption by endogenous chromophores (Oheim et al., 2001Oheim M. Beaurepaire E. Chaigneau E. Mertz J. Charpak S. Two-photon microscopy in brain tissue: parameters influencing the imaging depth.J. Neurosci. Methods. 2001; 111: 29-37Crossref PubMed Google Scholar, Svoboda and Block, 1994Svoboda K. Block S.M. Biological applications of optical forces.Annu. Rev. Biophys. Biomol. Struct. 1994; 23: 247-285Crossref PubMed Google Scholar, Yaroslavsky et al., 2002Yaroslavsky A.N. Schulze P.C. Yaroslavsky I.V. Schober R. Ulrich F. Schwarzmaier H.J. Optical properties of selected native and coagulated human brain tissues in vitro in the visible and near infrared spectral range.Phys. Med. Biol. 2002; 47: 2059-2073Crossref PubMed Scopus (179) Google Scholar). Second, because of the nature of nonlinear excitation, scattered excitation photons are too dilute to cause appreciable fluorescence (Figure 1B). Even deep in tissue, under conditions where most of the incidence photons are scattered, excitation is therefore still mostly limited to a small focal volume (but see the section on depth limitations). Third, because of localization of excitation, all fluorescence photons, ballistic and scattered, constitute useful signal if they are detected (Figure 1B). (In contrast, in wide-field and confocal microscopy, scattered fluorescence photons are either lost, or worse, contribute to background (Centonze and White, 1998Centonze V.E. White J.G. Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging.Biophys. J. 1998; 75: 2015-2024Abstract Full Text Full Text PDF PubMed Google Scholar)). Since in typical experiments in tissue the majority of fluorescence photons are scattered, this advantage of two-photon microscopy can be huge. Since localization of excitation is a direct consequence of the nonlinear nature of 2PE, other nonlinear contrast mechanisms (Mertz, 2004Mertz J. Nonlinear microscopy: new techniques and applications.Curr. Opin. Neurobiol. 2004; 14: 610-616Crossref PubMed Scopus (65) Google Scholar, Wilson and Sheppard, 1984Wilson T. Sheppard C. Theory and Practice of Scanning Optical Microscopy. Academic Press, New York1984Google Scholar), including 3PE of fluorescence (Maiti et al., 1997Maiti S. Shear J.B. Williams R.M. Zipfel W.R. Webb W.W. Measuring serotonin distribution in live cells with three-photon excitation.Science. 1997; 275: 530-532Crossref PubMed Scopus (288) Google Scholar) and second harmonic generation (Campagnola et al., 1999Campagnola P.J. Wei M.D. Lewis A. Loew L.M. High-resolution nonlinear optical imaging of live cells by second harmonic generation.Biophys. J. 1999; 77: 3341-3349Abstract Full Text Full Text PDF PubMed Google Scholar, Gannaway and Sheppard, 1978Gannaway J.N. Sheppard C.J.R. Second-harmonic imaging in the scanning optical microscope.Opt. Quant. Elect. 1978; 10: 435-439Crossref Scopus (139) Google Scholar), can be used for optical sectioning microscopy. 3PE may be useful to study endogenous tissue chromophores with one-photon spectra in the UV (Maiti et al., 1997Maiti S. Shear J.B. Williams R.M. Zipfel W.R. Webb W.W. Measuring serotonin distribution in live cells with three-photon excitation.Science. 1997; 275: 530-532Crossref PubMed Scopus (288) Google Scholar, Zipfel et al., 2003Zipfel W.R. Williams R.M. Webb W.W. Nonlinear magic: multiphoton microscopy in the biosciences.Nat. Biotechnol. 2003; 21: 1369-1377Crossref PubMed Scopus (1376) Google Scholar). Second harmonic generation can selectively excite probes that are aligned at interfaces and may therefore be useful to study the structure and function of membranes (Moreaux et al., 2000Moreaux L. Sandre O. Blanchard-Desce M. Mertz J. Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy.Opt. Lett. 2000; 25: 320-322Crossref PubMed Google Scholar, Dombeck et al., 2004Dombeck D.A. Blanchard-Desce M. Webb W.W. Optical recording of action potentials with second-harmonic generation microscopy.J. Neurosci. 2004; 24: 999-1003Crossref PubMed Scopus (113) Google Scholar). 2PE microscopy is typically implemented in a simple laser scanning microscope (Figure 1D). A laser is focused to a tight spot in the specimen plane and scanned in a raster over the sample. When the laser focus overlaps with fluorescent molecules in the sample, fluorescence photons are generated selectively in the tiny focal volume and detected by photodetectors. The signals are summed over pixel times (microseconds) and mapped to individual pixels of an image by the data acquisition computer. The simplicity of 2PE microscopes has allowed numerous labs to adapt confocal microscopes for 2PE microscopy (Majewska et al., 2000bMajewska A. Yiu G. Yuste R. A custom-made two-photon microscope and deconvolution system.Pflugers Arch. 2000; 441: 398-408Crossref PubMed Scopus (105) Google Scholar, Nikolenko et al., 2003Nikolenko V. Nemet B. Yuste R. A two-photon and second-harmonic microscope.Methods. 2003; 30: 3-15Crossref PubMed Scopus (53) Google Scholar, Ridsdale et al., 2004Ridsdale A. Micu I. Stys P.K. Conversion of the Nikon C1 confocal laser-scanning head for multiphoton excitation on an upright microscope.Appl. Opt. 2004; 43: 1669-1675Crossref PubMed Google Scholar). Other labs have opted for complete custom design (Mainen et al., 1999aMainen Z.F. Maletic-Savatic M. Shi S.H. Hayashi Y. Malinow R. Svoboda K. Two-photon imaging in living brain slices.Methods. 1999; 18: 231-239Crossref PubMed Scopus (106) Google Scholar, Pologruto et al., 2003Pologruto T.A. Sabatini B.L. Svoboda K. ScanImage: Flexible software for operating laser-scanning microscopes.Biomed. Eng. Online. 2003; 2: 13Crossref PubMed Scopus (205) Google Scholar, Tsai et al., 2002Tsai P.S. Nishimura N. Yoder E.J. White A. Dolnick E. Kleinfeld D. Principles, design and construction of a two photon scanning microscope for in vitro and in vivo studies.in: Frostig R. Methods for In Vivo Optical Imaging. CRC Press, Boca Raton, FL2002: 113-171Google Scholar). The principle differences between confocal and 2PE microscopes are the laser (see below) and the fluorescence detection path. In confocal microscopy, the epifluorescence light passes back through the scan mirrors and through a pinhole before detection (Conchello and Lichtman, 2005Conchello J.A. Lichtman J.W. Optical sectioning microscopy.Nat. Methods. 2005; 2: 920-931Crossref PubMed Scopus (233) Google Scholar). In 2PE microscopy, all fluorescence photons collected by the objective constitute useful signal, and the detector pinhole is not required. The optimal solution for the detection path is to project the objective back-aperture onto the photosensitive area of the photodetector (whole-field detection; Denk et al., 1995aDenk W. Piston D.W. Webb W.W. Two-photon molecular excitation in laser-scanning microscopy.in: Pawley J.B. Handbook of Biological Confocal Microscopy. Plenum Press, New York1995: 445-458Crossref Google Scholar, Mainen et al., 1999aMainen Z.F. Maletic-Savatic M. Shi S.H. Hayashi Y. Malinow R. Svoboda K. Two-photon imaging in living brain slices.Methods. 1999; 18: 231-239Crossref PubMed Scopus (106) Google Scholar, Tsai et al., 2002Tsai P.S. Nishimura N. Yoder E.J. White A. Dolnick E. Kleinfeld D. Principles, design and construction of a two photon scanning microscope for in vitro and in vivo studies.in: Frostig R. Methods for In Vivo Optical Imaging. CRC Press, Boca Raton, FL2002: 113-171Google Scholar) (Figure 1D). Since fluorescence photons may be scattered multiple times before exiting the tissue, they can appear to originate from a large effective field of view (Oheim et al., 2001Oheim M. Beaurepaire E. Chaigneau E. Mertz J. Charpak S. Two-photon microscopy in brain tissue: parameters influencing the imaging depth.J. Neurosci. Methods. 2001; 111: 29-37Crossref PubMed Google Scholar). Objectives with low magnifications (but maintaining high NA, e.g., 20× 0.9NA) are best for imaging in scattering tissue. However, the back-apertures of these objectives are large, and the scattered fluorescence light exiting the objective diverges rapidly (Oheim et al., 2001Oheim M. Beaurepaire E. Chaigneau E. Mertz J. Charpak S. Two-photon microscopy in brain tissue: parameters influencing the imaging depth.J. Neurosci. Methods. 2001; 111: 29-37Crossref PubMed Google Scholar). To collect as many fluorescence photons as possible, it is therefore necessary to bring large focusing elements close to the objective back-aperture, which is difficult to implement in commercial microscope stands. Similar considerations apply for transfluorescence collected through the condenser (Mainen et al., 1999aMainen Z.F. Maletic-Savatic M. Shi S.H. Hayashi Y. Malinow R. Svoboda K. Two-photon imaging in living brain slices.Methods. 1999; 18: 231-239Crossref PubMed Scopus (106) Google Scholar) (Figure 1D). Because two-photon cross-sections are small, very high instantaneous intensities of excitation light are required to generate sufficient signal levels. 2PE microscopy was made practical (Denk et al., 1990Denk W. Strickler J.H. Webb W.W. Two-photon laser scanning microscopy.Science. 1990; 248: 73-76Crossref PubMed Google Scholar) by the advent of lasers producing rapid trains of short pulses (Gosnell and Taylor, 1991Gosnell T.R. Taylor A.J. Selected Papers on Ultrafast Laser Technology. SPIE Press, Bellingham, WA1991Google Scholar). With the average power and pulse repetition rates constant, the 2PE efficiency increases as the inverse of the pulse duration. Mode-locked Ti:sapphire lasers have nearly ideal properties for 2PE microscopy. They produce a stream of pulses with repetition rates of ∼100 MHz; more than 100 pulses impinge on the sample within a typical pixel time, implying that variations in the number of pulses per pixel are small ( 1000 nm) (Zipfel et al., 2003Zipfel W.R. Williams R.M. Webb W.W. Nonlinear magic: multiphoton microscopy in the biosciences.Nat. Biotechnol. 2003; 21: 1369-1377Crossref PubMed Scopus (1376) Google Scholar). In this wavelength regime, other powerful light sources are available, for example mode-locked Ytterbium-doped lasers (Deguil et al., 2004Deguil N. Mottay E. Salin F. Legros P. Choquet D. Novel Diode-Pumped Infrared Tunable Laser System for Multi-Photon Microscopy.Microsc. Res. Tech. 2004; 63: 23-26Crossref PubMed Scopus (17) Google Scholar, Honninger et al., 1998Honninger C. Morier-Genoud F. Moser M. Keller U. Brovelli L.R. Harder C. Efficient and tunable diode-pumped femtosecond Yb: glass lasers.Opt. Lett. 1998; 23: 126-128Crossref PubMed Google Scholar) (fixed wavelength: ranging from 1030 nm to 1060 nm) or Cr:forsterite lasers (Chen et al., 2002aChen I.-H. Chu S.-W. Sun C.-K. Cheng P.-C. Lin B.-L. Wavelength dependent damage in biological multiphoton confocal microscopy: A micro-spectroscopic comparison between femtosecond Ti:sapphire and Cr:forsterite laser sources.Opt. Quantum Electron. 2002; 34: 1251-1266Crossref Scopus (56) Google Scholar) (tunable, 1200–1300 nm). The vast majority of laser scanning microscopes use galvanometer mirrors. They have excellent optical properties and allow zooming and image rotation. Their major drawback is their relatively slow speed (>1 ms per line); a typical image requires ∼1 s. Since many neurophysiological processes occur over milliseconds, faster scanning methods are needed. In multifocal scanning, the beam is divided into beamlets that are simultaneously scanned across the sample (Andresen et al., 2001Andresen V. Egner A. Hell S.W. Time-multiplexed multifocal multiphoton microscope.Opt. Lett. 2001; 26: 75-77Crossref PubMed Google Scholar, Bewersdorf et al., 1998Bewersdorf J. Pick R. Hell S.W. Multifocal multiphoton microscopy.Opt. Lett. 1998; 23: 655-657Crossref PubMed Google Scholar, Kurtz et al., 2006Kurtz R. Fricke M. Kalb J. Tinnefeld P. Sauer M. Application of multiline two-photon microscopy to functional in vivo imaging.J. Neurosci. Methods. 2006; 151: 276-286Crossref PubMed Scopus (39) Google Scholar). This approach demands imaging detectors (i.e., CCDs) since the fluorescence excited by different beamlets needs to be distinguished. In scattering samples, the image will be blurred. In addition, dividing the beam into beamlets with lower power reduces nonlinear excitation. In highly scattering tissue, point scanning with whole-field detection is preferable. Rapid raster scanning can be performed with rotating polygonal mirrors (Kim et al., 1999
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