Multiphoton microscopy (MPM) has found a niche in the world of biological imaging as the best noninvasive means of fluorescence microscopy in tissue explants and living animals. Coupled with transgenic mouse models of disease and 'smart' genetically encoded fluorescent indicators, its use is now increasing exponentially. Properly applied, it is capable of measuring calcium transients 500 μm deep in a mouse brain, or quantifying blood flow by imaging shadows of blood cells as they race through capillaries. With the multitude of possibilities afforded by variations of nonlinear optics and localized photochemistry, it is possible to image collagen fibrils directly within tissue through nonlinear scattering, or release caged compounds in sub-femtoliter volumes.
This is a preview of subscription content, access via your institution
Open Access articles citing this article.
Contribution of autofluorescence from intracellular proteins in multiphoton fluorescence lifetime imaging
Scientific Reports Open Access 05 October 2022
Nondestructive circadian profiling of starch content in fresh intact Arabidopsis leaf with two-photon fluorescence and second-harmonic generation imaging
Scientific Reports Open Access 03 October 2022
Scientific Reports Open Access 01 September 2022
Subscribe to Journal
Get full journal access for 1 year
only $8.25 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Get time limited or full article access on ReadCube.
All prices are NET prices.
Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).
Yuste, R. & Denk, W. Dendritic spines as basic functional units of neuronal integration. Nature 375, 682–684 (1995).
Mainen, Z.F., Malinow, R. & Svoboda, K. Synaptic calcium transients in single spines indicate that NMDA receptors are not saturated. Nature 399, 151–155 (1999).
Rose, C.R., Kovalchuk, Y., Eilers, J. & Konnerth, A. Two-photon Na+ imaging in spines and fine dendrites of central neurons. Pflugers Arch. 439, 201–207 (1999).
Tan, Y.P. & Llano, I. Modulation by K+ channels of action potential-evoked intracellular Ca2+ concentration rises in rat cerebellar basket cell axons. J. Physiol. 520 Pt 1, 65–78 (1999).
Cox, C.L., Denk, W., Tank, D.W. & Svoboda, K. Action potentials reliably invade axonal arbors of rat neocortical neurons. Proc. Natl. Acad. Sci. USA 97, 9724–9728 (2000).
Majewska, A., Tashiro, A. & Yuste, R. Regulation of spine calcium dynamics by rapid spine motility. J. Neurosci. 20, 8262–8268 (2000).
Oertner, T.G. Functional imaging of single synapses in brain slices. Exp. Physiol. 87, 733–736 (2002).
Frick, A., Magee, J., Koester, H.J., Migliore, M. & Johnston, D. Normalization of Ca2+ signals by small oblique dendrites of CA1 pyramidal neurons. J. Neurosci. 23, 3243–3250 (2003).
Lendvai, B., Zelles, T., Rozsa, B. & Vizi, E.S. A vinca alkaloid enhances morphological dynamics of dendritic spines of neocortical layer 2/3 pyramidal cells. Brain Res. Bull. 59, 257–260 (2003).
Sabatini, B.L. & Svoboda, K. Analysis of calcium channels in single spines using optical fluctuation analysis. Nature 408, 589–593 (2000).
Svoboda, K., Denk, W., Kleinfeld, D. & Tank, D.W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997).
Helmchen, F., Svoboda, K., Denk, W. & Tank, D.W. In vivo dendritic calcium dynamics in deep-layer cortical pyramidal neurons. Nat. Neurosci. 2, 989–996 (1999).
Stosiek, C., Garaschuk, O., Holthoff, K. & Konnerth, A. In vivo two-photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. USA 100, 7319–7324 (2003).
Helmchen, F. & Waters, J. Ca(2+) imaging in the mammalian brain in vivo. Eur. J. Pharmacol. 447, 119–129 (2002).
Svoboda, K., Tank, D.W. & Denk, W. Direct measurement of coupling between dendritic spines and shafts. Science 272, 716–719 (1996).
Ladewig, T. et al. Spatial profiles of store-dependent calcium release in motoneurones of the nucleus hypoglossus from newborn mouse. J. Physiol. 547, 775–787 (2003).
Christie, R.H. et al. Growth arrest of individual senile plaques in a model of Alzheimer's disease observed by in vivo multiphoton microscopy. J. Neurosci. 21, 858–864 (2001).
Bacskai, B.J. et al. Non-Fc-mediated mechanisms are involved in clearance of amyloid-beta in vivo by immunotherapy. J. Neurosci. 22, 7873–7878 (2002).
D'Amore, J.D. et al. In vivo multiphoton imaging of a transgenic mouse model of Alzheimer disease reveals marked thioflavine-S-associated alterations in neurite trajectories. J. Neuropathol. Exp. Neurol. 62, 137–145 (2003).
Bacskai, B.J. et al. Imaging of amyloid-beta deposits in brains of living mice permits direct observation of clearance of plaques with immunotherapy. Nat. Med. 7, 369–372 (2001).
Brown, E.B. et al. In vivo measurement of gene expression, angiogenesis and physiological function in tumors using multiphoton laser scanning microscopy. Nat. Med. 7, 864–868 (2001).
McDonald, D.M. & Choyke, P.L. Imaging of angiogenesis: from microscope to clinic. Nat. Med. 9, 713–725 (2003).
Wang, W. et al. Single cell behavior in metastatic primary mammary tumors correlated with gene expression patterns revealed by molecular profiling. Cancer Res. 62, 6278–6288 (2002).
Wolf, K. et al. Compensation mechanism in tumor cell migration: mesenchymal-amoeboid transition after blocking of pericellular proteolysis. J. Cell Biol. 160, 267–277 (2003).
Cahalan, M.D., Parker, I., Wei, S.H. & Miller, M.J. Two-photon tissue imaging: seeing the immune system in a fresh light. Nat. Rev. Immunol. 2, 872–880 (2002).
Miller, M.J., Wei, S.H., Parker, I. & Cahalan, M.D. Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296, 1869–1873 (2002).
Wei, S.H., Miller, M.J., Cahalan, M.D. & Parker, I. Two-photon imaging in intact lymphoid tissue. Adv. Exp. Med. Biol. 512, 203–208 (2002).
Miller, M.J., Wei, S.H., Cahalan, M.D. & Parker, I. Autonomous T cell trafficking examined in vivo with intravital two-photon microscopy. Proc. Natl. Acad. Sci. USA 100, 2604–2609 (2003).
Acuto, O. T cell–dendritic cell interaction in vivo: random encounters favor development of long-lasting ties. Science STKE 2003, PE28 (2003).
Squirrell, J.M., Wokosin, D.L., White, J.G. & Bavister, B.D. Long-term two-photon fluorescence imaging of mammalian embryos without compromising viability. Nat. Biotechnol. 17, 763–767 (1999).
Gryczynski, I., Szmacinski, H. & Lakowicz, J.R. On the possibility of calcium imaging using Indo-1 with three-photon excitation. Photochem. Photobiol. 62, 804–808 (1995).
Lakowicz, J.R. et al. Time-resolved fluorescence spectroscopy and imaging of DNA labeled with DAPI and Hoechst 33342 using three-photon excitation. Biophys. J. 72, 567–578 (1997).
Maiti, S., Shear, J.B., Williams, R.M., Zipfel, W.R. & Webb, W.W. Measuring serotonin distribution in live cells with three-photon excitation. Science 275, 530–532 (1997).
Williams, R.M., Shear, J.B., Zipfel, W.R., Maiti, S. & Webb, W.W. Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence. Biophys. J. 76, 1835–1846 (1999).
Xu, C., Zipfel, W., Shear, J.B., Williams, R.M. & Webb, W.W. Multiphoton fluorescence excitation: new spectral windows for biological nonlinear microscopy. Proc. Natl. Acad. Sci. USA 93, 10763–10768 (1996).
Zipfel, W.R. et al. Live tissue intrinsic emission microscopy using multiphoton-excited native fluorescence and second harmonic generation. Proc. Natl. Acad. Sci. USA 100, 7075–7080 (2003).
Freund, I. & Deutsch, M. 2nd-harmonic microscopy of biological tissue. Opt. Lett. 11, 94–96 (1986).
Campagnola, P.J., Clark, H.A., Mohler, W.A., Lewis, A. & Loew, L.M. Second-harmonic imaging microscopy of living cells. J. Biomed. Opt. 6, 277–286 (2001).
Mertz, J. & Moreaux, L. Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers. Opt. Commun. 196, 325–330 (2001).
Moreaux, L., Sandre, O., Charpak, S., Blanchard–Desce, M. & Mertz, J. Coherent scattering in multi-harmonic light microscopy. Biophys. J. 80, 1568–1574 (2001).
Campagnola, P.J. et al. Three-dimensional high-resolution second-harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys. J. 82, 493–508 (2002).
Campagnola, P.J., Mohler, W. & Millard, A.E. 3-dimensional high-resolution second harmonic generation imaging of endogenous structural proteins in biological tissues. Biophys. J. 82, 175a–175a (2002).
Dombeck, D.A. et al. Uniform polarity microtubule assemblies imaged in native brain tissue by second-harmonic generation microscopy. Proc. Natl. Acad. Sci. USA 100, 7081–7086 (2003).
Zoumi, A., Yeh, A. & Tromberg, B.J. Imaging cells and extracellular matrix in vivo by using second-harmonic generation and two-photon excited fluorescence. Proc. Natl. Acad. Sci. USA 99, 11014–11019 (2002).
Barad, Y., Eisenberg, H., Horowitz, M. & Silberberg, Y. Nonlinear scanning laser microscopy by third harmonic generation. Appl. Phys. Lett. 70, 922–924 (1997).
Muller, M., Squier, J., Wilson, K.R. & Brakenhoff, G.J. 3D microscopy of transparent objects using third-harmonic generation. J. Microsc. 191, 266–274 (1998).
Yelin, D., Oron, D., Korkotian, E., Segal, M. & Silbergerg, Y. Third-harmonic microscopy with a titanium-sapphire laser. Appl. Phys. B–Lasers O 74, S97–S101 (2002).
Sheppard, C.J.R. & Kompfner, R. Resonant scanning optical microscope. Appl. Optics 17, 2879–2882 (1978).
Duncan, M.D., Reintjes, J. & Manuccia, T.J. Scanning coherent anti-Stokes Raman microscope. Opt. Lett. 7, 350–352 (1982).
Zumbusch, A., Holtom, G.R. & Xie, X.S. Vibrational mircoscopy using coherent anti-Stokes Raman scattering (1999). Phys. Rev. Lett. 82, 4014–4017 (1999).
Muller, M., Squier, J., De Lange, C.A. & Brakenhoff, G.J. CARS microscopy with folded BoxCARS phasematching. J. Microsc. 197 (Pt 2), 150–158 (2000).
Piston, D.W., Summers, R.G., Knobel, S.M. & Morrill, J.B. Characterization of involution during sea urchin gastrulation using two-photon excited photorelease and confocal microscopy. Microsc. Microanal. 4, 404–414 (1998).
Furuta, T. et al. Brominated 7-hydroxycoumarin-4-ylmethyls: photolabile protecting groups with biologically useful cross-sections for two photon photolysis. Proc. Natl. Acad. Sci. USA 96, 1193–1200 (1999).
Matsuzaki, M. et al. Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat. Neurosci. 4, 1086–1092 (2001).
Echevarria, W., Leite, M.F., Guerra, M.T., Zipfel, W.R. & Nathanson, M.H. Regulation of calcium signals in the nucleus by a nucleoplasmic reticulum. Nat. Cell. Biol. 5, 440–446 (2003).
Berland, K.M., So, P.T. & Gratton, E. Two-photon fluorescence correlation spectroscopy: method and application to the intracellular environment. Biophys. J. 68, 694–701 (1995).
Schwille, P., Haupts, U., Maiti, S. & Webb, W.W. Molecular dynamics in living cells observed by fluorescence correlation spectroscopy with one- and two-photon excitation. Biophys. J. 77, 2251–2265 (1999).
Brown, E.B., Wu, E.S., Zipfel, W. & Webb, W.W. Measurement of molecular diffusion in solution by multiphoton fluorescence photobleaching recovery. Biophys. J. 77, 2837–2849 (1999).
Zipfel, W.R. & Webb, W.W. In vivo diffusion measurements using multiphoton-excited fluorescence photobleaching recovery (MPFPR) and fluorescence correlation spectroscopy (MPFCS) in Methods in Cellular Imaging (ed. Periasamy, A.) 345–376 (Oxford University Press, Oxford, UK, 2001).
Stroh, M., Zipfel, W.R., Williams, R.M., Webb, W.W. & Saltzman, W.M. Diffusion of nerve growth factor in rat striatum as determined by multiphoton microscopy. Biophys. J. 85, 581–588 (2003).
Heinze, K.G., Koltermann, A. & Schwille, P. Simultaneous two-photon excitation of distinct labels for dual-color fluorescence cross correlation analysis. Proc. Natl. Acad. Sci. USA 97, 10377–10382 (2000).
Tirlapur, U.K. & Konig, K. Targeted transfection by femtosecond laser. Nature 418, 290–291 (2002).
Konig, K., Riemann, I. & Fritzsche, W. Nanodissection of human chromosomes with near-infrared femtosecond laser pulses. Opt. Lett. 26, 819–821 (2001).
Göppert-Mayer, M. Uber elementarakte mit zwei quantensprüngen. Ann. Phys. 9, 273–294 (1931).
Xu, C. & Webb, W.W. Multiphoton excitation of molecular fluorophores and nonlinear laser microscopy in Topics in Fluorescence Spectroscopy: Volume 5: Nonlinear and Two-Photon-Induced Fluorescence. (ed. Lakowicz, J.) 471–540 (Plenum Press, New York, 1997).
Xu, C. & Webb, W.W. Measurement of two-photon excitation cross sections of molecular fluorophores with data from 690 nm to 1050 nm. J. Opt. Soc. Am. B 13, 481–491 (1996).
Steinfeld, J.I. Molecules and Radiation. (MIT Press, Cambridge, MA, 1989).
Huang, S., Heikal, A.A. & Webb, W.W. Two-photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys. J. 82, 2811–2825 (2002).
Piston, D.W., Masters, B.R. & Webb, W.W. Three-dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two-photon excitation laser scanning microscopy. J. Microsc. 178 (Pt 1), 20–27 (1995).
Wong, B.J., Wallace, V., Coleno, M., Benton, H.P. & Tromberg, B.J. Two-photon excitation laser scanning microscopy of human, porcine, and rabbit nasal septal cartilage. Tissue Eng. 7, 599–606 (2001).
Noda, M. et al. Switch to anaerobic glucose metabolism with NADH accumulation in the beta-cell model of mitochondrial diabetes. Characteristics of betaHC9 cells deficient in mitochondrial DNA transcription. J. Biol. Chem. 277, 41817–41826 (2002).
Zhang, Q., Piston, D.W. & Goodman, R.H. Regulation of corepressor function by nuclear NADH. Science 295, 1895–1897 (2002).
Larson, D.R. et al. Water-soluble quantum dots for multiphoton fluorescence imaging in vivo. Science 300, 1434–1436 (2003).
Albota, M. et al. Design of organic molecules with large two-photon absorption cross sections. Science 281, 1653–1656 (1998).
Wang, X.M. et al. Synthesis of new symmetrically substituted stilbenes with large multi-photon absorption cross section and strong two-photon–induced blue fluorescence. Bull. Chem. Soc. Jpn 74, 1977–1982 (2001).
Zhou, X. et al. One- and two-photon absorption properties of novel multi-branched molecules. Phys. Chem. Chem. Phys. 4, 4346–4352 (2002).
Heikal, A.A., Hess, S.T. & Webb, W.W. Multiphoton molecular spectroscopy and excited-state dynamics of enhanced green fluorescent protein (EGFP): acid-base specificity. Chem. Phys. 274, 37–55 (2001).
Blab, G.A., Lommerse, P.H.M., Cognet, L., Harms, G.S. & Schmidt, T. Two-photon excitation action cross-sections of the autofluorescent proteins. Chem. Phys. Lett. 350, 71–77 (2001).
Hanson, G.T. et al. Green fluorescent protein variants as ratiometric dual emission pH sensors. 1. Structural characterization and preliminary application. Biochemistry 41, 15477–15488 (2002).
Tsai, P.S. et al. All-optical histology using ultrashort laser pulses. Neuron 39, 27–41 (2003).
Mainen, Z.F. et al. Two-photon imaging in living brain slices. Methods 18, 231–239, (1999).
Shi, S.H. et al. Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 284, 1811–1816 (1999).
D'Apuzzo, M., Mandolesi, G., Reis, G. & Schuman, E.M. Abundant GFP expression and LTP in hippocampal acute slices by in vivo injection of Sindbis virus. J. Neurophysiol. 86, 1037–1042 (2001).
Potter, S.M. et al. Structure and emergence of specific olfactory glomeruli in the mouse. J. Neurosci. 21, 9713–9723 (2001).
Strome, S. et al. Spindle dynamics and the role of gamma-tubulin in early Caenorhabditis elegans embryos. Mol. Biol. Cell 12, 1751–1764 (2001).
Ahmed, F. et al. GFP expression in the mammary gland for imaging of mammary tumor cells in transgenic mice. Cancer Res. 62, 7166–7169 (2002).
Lawson, N.D. & Weinstein, B.M. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318 (2002).
Bestvater, F. et al. Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging. J. Microsc. 208, 108–115 (2002).
Dickinson, M.E., Simbuerger, E., Zimmermann, B., Waters, C.W. & Fraser, S.E. Multiphoton excitation spectra in biological samples. J. Biomed. Opt. 8, 329–338 (2003).
Periasamy, A. Fluorescence resonance energy transfer microscopy: a mini review. J. Biomed. Opt. 6, 287–291 (2001).
Majoul, I., Straub, M., Duden, R., Hell, S.W. & Soling, H.D. Fluorescence resonance energy transfer analysis of protein-protein interactions in single living cells by multifocal multiphoton microscopy. J. Biotechnol. 82, 267–277 (2002).
Bacskai, B.J., Skoch, J., Hickey, G.A., Allen, R. & Hyman, B.T. Fluorescence resonance energy transfer determinations using multiphoton fluorescence lifetime imaging microscopy to characterize amyloid-beta plaques. J. Biomed. Opt. 8, 368–375 (2003).
Gu, M. & Sheppard, C.J.R. Comparison of three-dimensional imaging properties between two-photon and single-photon fluorescence microscopy. J. Microsc. 177, 128–137 (1995).
Centonze, V.E. & White, J.G. Multiphoton excitation provides optical sections from deeper within scattering specimens than confocal imaging. Biophys. J. 75, 2015–2024 (1998).
Periasamy, A., Skoglund, P., Noakes, C. & Keller, R. An evaluation of two-photon excitation versus confocal and digital deconvolution fluorescence microscopy imaging in Xenopus morphogenesis. Microsc. Res. Technol. 47, 172–181 (1999).
Schilders, S.P. & Gu, M. Limiting factors on image quality in imaging through turbid media under single-photon and two-photon excitation. Microsc. Microanal. 6, 156–160 (2000).
Sheppard, C.J.R. & Gu, M. Image-formation in 2-photon fluorescence microscopy. Optik 86, 104–106 (1990).
Richards, B. & Wolf, E. Electromagnetic Diffraction in Optical Systems. 2. Structure of the Image Field in an Aplanatic System. Proc. R. Soc. Lon. Ser. –A 253, 358–379 (1959).
Sheppard, C.J.R. & Matthews, H.J. Imaging in high-aperture optical systems. J. Opt. Soc. Am. A 4, 1354–1360 (1987).
Beaurepaire, E., Oheim, M. & Mertz, J. Ultra-deep two-photon fluorescence excitation in turbid media. Opt. Commun. 188, 25–29 (2001).
Theer, P., Hasan, M.T. & Denk, W. Two-photon imaging to a depth of 1000 microm in living brains by use of a Ti:Al2O3 regenerative amplifier. Opt. Lett. 28, 1022–1024 (2003).
Curley, P.F., Ferguson, A.I., White, J.G. & Amos, W.B. Application of a femtosecond self-sustaining mode-locked Ti:sapphire laser to the field of laser scanning confocal microscopy. Opt. Quant. Electron. 24, 851–859 (1992).
Hockberger, P.E. et al. Activation of flavin-containing oxidases underlies light-induced production of H2O2 in mammalian cells. Proc. Natl. Acad. Sci. USA 96, 6255–6260 (1999).
Wokosin, D.L., Squirrell, J.M., Eliceiri, K.W. & White, J.G. Optical workstation with concurrent, independent multiphoton imaging and experimental laser microbeam capabilities. Rev. Sci. Instrum. 74, 193–201 (2003).
Hopkins, J. & Sibbett, W. Ultrashort lasers: big payoff in a flash. Sci. Am. 283, 73–79 (2000).
Soeller, C. & Cannell, M.B. Construction of a two-photon microscope and optimization of illumination pulse duration. Pflugers Arch. 432, 555–561 (1996).
Squier, J. & Muller, M. High resolution nonlinear microscopy: A review of sources and methods for achieving optimal imaging. Rev. Sci. Instrum. 72, 2855–2867 (2001).
Muller, D., Squier, J. & Brakenhoff, G.J. Measurement of femtosecond pulses in the focal point of a high-numerical-aperture lens by two-photon absorption. Opt. Lett. 20, 1038–1040 (1995).
Guild, J.B., Xu, C. & Webb, W.W. Measurement of group delay dispersion of high numerical aperture objective lenses using two-photon excited fluorescence. Appl. Optics 36, 397–401 (1997).
Muller, M., Squier, J., Wolleschensky, R., Simon, U. & Brakenhoff, G.J. Dispersion pre-compensation of 15 femtosecond optical pulses for high-numerical-aperture objectives. J. Microsc. 191, 141–150 (1998).
Majewska, A., Yiu, G. & Yuste, R. A custom-made two-photon microscope and deconvolution system. Pflugers Arch. 441, 398–408 (2000).
Tsai, P.S. et al. Principles, design and construction of a two photon scanning microscope for in vitro and in vivo studies in Methods for In Vivo Optical Imaging (ed. Frostig, R.) 113–171 (CRC Press, Boca Raton, FL, 2002).
Iyer, V., Losavio, B.E. & Saggau, P. Compensation of spatial and temporal dispersion for acousto-optic multiphoton laser-scanning microscopy. J. Biomed. Opt. 8, 460–471 (2003).
Pawley, J.B. Handbook of Biological Confocal Microscopy, edn 2. (Plenum Press, New York, 1995).
Fan, G.Y. et al. Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys. J. 76, 2412–2420 (1999).
Nguyen, Q.T., Callamaras, N., Hsieh, C. & Parker, I. Construction of a two-photon microscope for video-rate Ca(2+) imaging. Cell Calcium 30, 383–393 (2001).
Gauderon, R., Lukins, P.B. & Sheppard, C.J. Effect of a confocal pinhole in two-photon microscopy. Microsc. Res. Technol. 47, 210–214 (1999).
Oheim, M., Beaurepaire, E., Chaigneau, E., Mertz, J. & Charpak, S. Two-photon microscopy in brain tissue: parameters influencing the imaging depth. J. Neurosci. Methods 111, 29–37 (2001).
Egner, A., Jakobs, S. & Hell, S.W. Fast 100-nm resolution three-dimensional microscope reveals structural plasticity of mitochondria in live yeast. Proc. Natl. Acad. Sci. USA 99, 3370–3375 (2002).
Tan, Y.P., Llano, I., Hopt, A., Wurriehausen, F. & Neher, E. Fast scanning and efficient photodetection in a simple two-photon microscope. J. Neurosci. Methods 92, 123–135 (1999).
Gratton, E., Breusegem, S., Sutin, J., Ruan, Q. & Barry, N. Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods. J. Biomed. Opt. 8, 381–390 (2003).
Moreaux, L., Sandre, O. & Mertz, J. Membrane imaging by second-harmonic generation microscopy. J. Opt. Soc. Am. B 17, 1685–1694 (2000).
Peleg, G., Lewis, A., Linial, M. & Loew, L.M. Nonlinear optical measurement of membrane potential around single molecules at selected cellular sites. Proc. Natl. Acad. Sci. USA 96, 6700–6704 (1999).
Moreaux, L., Sandre, O., Blanchard–Desce, M. & Mertz, J. Membrane imaging by simultaneous second-harmonic generation and two-photon microscopy. Opt. Lett. 25, 320–322 (2000).
Millard, A.C., Jin, L., Lewis, A. & Loew, L.M. Direct measurement of the voltage sensitivity of second-harmonic generation from a membrane dye in patch-clamped cells. Opt. Lett. 28, 1221–1223 (2003).
Mohler, W., Millard, A.C. & Campagnola, P.J. Second harmonic generation imaging of endogenous structural proteins. Methods 29, 97–109 (2003).
Konig, K., So, P.T., Mantulin, W.W., Tromberg, B.J. & Gratton, E. Two-photon excited lifetime imaging of autofluorescence in cells during UVA and NIR photostress. J. Microsc. 183 (Pt 3), 197–204 (1996).
Koester, H.J., Baur, D., Uhl, R. & Hell, S.W. Ca2+ fluorescence imaging with pico– and femtosecond two-photon excitation: signal and photodamage. Biophys. J. 77, 2226–2236 (1999).
Hopt, A. & Neher, E. Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys. J. 80, 2029–2036 (2001).
Dittrich, P.S. & Schwille, P. Photobleaching and stabilization of fluorophores used for single-molecule analysis with one- and two-photon excitation. Appl. Phys. B. Lasers O 73, 829–837 (2001).
Patterson, G.H. & Piston, D.W. Photobleaching in two-photon excitation microscopy. Biophys. J. 78, 2159–2162 (2000).
Neil, M.A. et al. Adaptive aberration correction in a two-photon microscope. J. Microsc. 200 (Pt 2), 105–108 (2000).
Booth, M.J., Neil, M.A. & Wilson, T. New modal wave-front sensor: application to adaptive confocal fluorescence microscopy and two-photon excitation fluorescence microscopy. J. Opt. Soc. Am. A Opt. Image Sci. Vis. 19, 2112–2120 (2002).
Marsh, P.N., Burns, D. & Girkin, J.M. Practical implementation of adaptive optics in multiphoton microscopy. Opt. Express 11, 1123–1130 (2003).
Brunner, F. et al. Diode-pumped femtosecond Yb:KGd(WO/sub 4/)/sub 2/ laser with 1.1-W average power. Opt. Lett. 25, 1119–1121 (2000).
Ilday, F.O., Lim, H., Buckley, J.R. & Wise, F.W. Practical all-fiber source of high-power, 120-fs pulses at 1 micron. Opt. Lett. 28, 1362–1364 (2003).
Jung, J.C. & Schnitzer, M.J. Multiphoton endoscopy. Opt. Lett. 28, 902–904 (2003).
Bird, D. & Gu, M. Two-photon fluorescence endoscopy with a mirco-optic scanning head. Opt. Lett. 28, 1552–1554 (2003).
Ouzounov, D.G. et al. Delivery of nanojoule femtosecond pulses through large-core microstructured fibers. Opt. Lett. 27, 1513–1515 (2002).
Pastirk, I., Dela Cruz, J.M., Walowicz, K.A., Lozovoy, V.V. & Dantus, M. Selective two-photon microscopy with shaped femtosecond pulses. Opt. Express 11, 1695–1701 (2003).
Williams, R.M. & Webb, W.W. Single granule pH cycling in antigen-induced mast cell secretion. J. Cell Sci. 113 (Pt 21), 3839–3850 (2000).
Kloppenburg, P., Zipfel, W.R., Webb, W.W. & Harris–Warrick, R.M. Highly localized Ca(2+) accumulation revealed by multiphoton microscopy in an identified motoneuron and its modulation by dopamine. J. Neurosci. 20, 2523–2533 (2000).
Kleinfeld, 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 95, 15741–15746 (1998).
Cornell Research Foundation holds the patent on Multiphoton Microscopy and the authors may benefit from its licenses.
About this article
Cite this article
Zipfel, W., Williams, R. & Webb, W. Nonlinear magic: multiphoton microscopy in the biosciences. Nat Biotechnol 21, 1369–1377 (2003). https://doi.org/10.1038/nbt899
This article is cited by
Adaptive optical microscopy via virtual-imaging-assisted wavefront sensing for high-resolution tissue imaging
Nature Physics (2022)
Nature Protocols (2022)
Scientific Reports (2022)