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Nonlinear magic: multiphoton microscopy in the biosciences

Nature Biotechnology volume 21, pages 1369–1377 (2003)Cite this article

Abstract

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.

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Figure 1: Publications employing, developing or reviewing MPM (from PubMed and ISI).
Figure 2: Localization of excitation by two-photon excitation.
Figure 3: Two-photon action cross-sections.
Figure 4: The two-photon excitation volume.
Figure 5: Components of a multiphoton microscope.
Figure 6: Applications showing various capabilities of MPM.
Figure 7: Two-photon fluorescence and SHG.

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References

  1. Denk, W., Strickler, J.H. & Webb, W.W. Two-photon laser scanning fluorescence microscopy. Science 248, 73–76 (1990).

    CAS  PubMed  Google Scholar 

  2. Yuste, R. & Denk, W. Dendritic spines as basic functional units of neuronal integration. Nature 375, 682–684 (1995).

    CAS  PubMed  Google Scholar 

  3. 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).

    CAS  PubMed  Google Scholar 

  4. 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).

    CAS  PubMed  Google Scholar 

  5. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  6. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  7. Majewska, A., Tashiro, A. & Yuste, R. Regulation of spine calcium dynamics by rapid spine motility. J. Neurosci. 20, 8262–8268 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  8. Oertner, T.G. Functional imaging of single synapses in brain slices. Exp. Physiol. 87, 733–736 (2002).

    PubMed  Google Scholar 

  9. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  10. 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).

    CAS  PubMed  Google Scholar 

  11. Sabatini, B.L. & Svoboda, K. Analysis of calcium channels in single spines using optical fluctuation analysis. Nature 408, 589–593 (2000).

    CAS  PubMed  Google Scholar 

  12. Svoboda, K., Denk, W., Kleinfeld, D. & Tank, D.W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature 385, 161–165 (1997).

    CAS  PubMed  Google Scholar 

  13. 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).

    CAS  PubMed  Google Scholar 

  14. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  15. Helmchen, F. & Waters, J. Ca(2+) imaging in the mammalian brain in vivo. Eur. J. Pharmacol. 447, 119–129 (2002).

    CAS  PubMed  Google Scholar 

  16. Svoboda, K., Tank, D.W. & Denk, W. Direct measurement of coupling between dendritic spines and shafts. Science 272, 716–719 (1996).

    CAS  PubMed  Google Scholar 

  17. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  18. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  19. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  20. 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).

    CAS  PubMed  Google Scholar 

  21. 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).

    CAS  PubMed  Google Scholar 

  22. 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).

    CAS  PubMed  Google Scholar 

  23. McDonald, D.M. & Choyke, P.L. Imaging of angiogenesis: from microscope to clinic. Nat. Med. 9, 713–725 (2003).

    CAS  PubMed  Google Scholar 

  24. 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).

    CAS  PubMed  Google Scholar 

  25. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  26. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  27. 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).

    CAS  PubMed  Google Scholar 

  28. 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).

    PubMed  Google Scholar 

  29. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  30. Acuto, O. T cell–dendritic cell interaction in vivo: random encounters favor development of long-lasting ties. Science STKE 2003, PE28 (2003).

    Google Scholar 

  31. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 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).

    CAS  PubMed  Google Scholar 

  33. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 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).

    CAS  PubMed  Google Scholar 

  35. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. Freund, I. & Deutsch, M. 2nd-harmonic microscopy of biological tissue. Opt. Lett. 11, 94–96 (1986).

    CAS  PubMed  Google Scholar 

  39. 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).

    CAS  PubMed  Google Scholar 

  40. Mertz, J. & Moreaux, L. Second-harmonic generation by focused excitation of inhomogeneously distributed scatterers. Opt. Commun. 196, 325–330 (2001).

    CAS  Google Scholar 

  41. Moreaux, L., Sandre, O., Charpak, S., Blanchard–Desce, M. & Mertz, J. Coherent scattering in multi-harmonic light microscopy. Biophys. J. 80, 1568–1574 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  43. 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).

    Google Scholar 

  44. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  46. Barad, Y., Eisenberg, H., Horowitz, M. & Silberberg, Y. Nonlinear scanning laser microscopy by third harmonic generation. Appl. Phys. Lett. 70, 922–924 (1997).

    CAS  Google Scholar 

  47. 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).

    CAS  PubMed  Google Scholar 

  48. 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).

    CAS  Google Scholar 

  49. Sheppard, C.J.R. & Kompfner, R. Resonant scanning optical microscope. Appl. Optics 17, 2879–2882 (1978).

    CAS  Google Scholar 

  50. Duncan, M.D., Reintjes, J. & Manuccia, T.J. Scanning coherent anti-Stokes Raman microscope. Opt. Lett. 7, 350–352 (1982).

    CAS  PubMed  Google Scholar 

  51. Zumbusch, A., Holtom, G.R. & Xie, X.S. Vibrational mircoscopy using coherent anti-Stokes Raman scattering (1999). Phys. Rev. Lett. 82, 4014–4017 (1999).

    Google Scholar 

  52. 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).

    PubMed  Google Scholar 

  53. 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).

    CAS  PubMed  Google Scholar 

  54. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Matsuzaki, M. et al. Dendritic spine geometry is critical for AMPA receptor expression in hippocampal CA1 pyramidal neurons. Nat. Neurosci. 4, 1086–1092 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  56. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  58. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  59. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  60. 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).

    Google Scholar 

  61. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  62. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  63. Tirlapur, U.K. & Konig, K. Targeted transfection by femtosecond laser. Nature 418, 290–291 (2002).

    CAS  PubMed  Google Scholar 

  64. Konig, K., Riemann, I. & Fritzsche, W. Nanodissection of human chromosomes with near-infrared femtosecond laser pulses. Opt. Lett. 26, 819–821 (2001).

    CAS  PubMed  Google Scholar 

  65. Göppert-Mayer, M. Uber elementarakte mit zwei quantensprüngen. Ann. Phys. 9, 273–294 (1931).

    Google Scholar 

  66. 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).

    Google Scholar 

  67. 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).

    CAS  Google Scholar 

  68. Steinfeld, J.I. Molecules and Radiation. (MIT Press, Cambridge, MA, 1989).

    Google Scholar 

  69. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  70. 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).

    CAS  PubMed  Google Scholar 

  71. 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).

    CAS  PubMed  Google Scholar 

  72. 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).

    CAS  PubMed  Google Scholar 

  73. Zhang, Q., Piston, D.W. & Goodman, R.H. Regulation of corepressor function by nuclear NADH. Science 295, 1895–1897 (2002).

    CAS  PubMed  Google Scholar 

  74. Larson, D.R. et al. Water-soluble quantum dots for multiphoton fluorescence imaging in vivo. Science 300, 1434–1436 (2003).

    CAS  PubMed  Google Scholar 

  75. Albota, M. et al. Design of organic molecules with large two-photon absorption cross sections. Science 281, 1653–1656 (1998).

    CAS  PubMed  Google Scholar 

  76. 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).

    CAS  Google Scholar 

  77. Zhou, X. et al. One- and two-photon absorption properties of novel multi-branched molecules. Phys. Chem. Chem. Phys. 4, 4346–4352 (2002).

    CAS  Google Scholar 

  78. 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).

    CAS  Google Scholar 

  79. 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).

    CAS  Google Scholar 

  80. 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).

    CAS  PubMed  Google Scholar 

  81. Tsai, P.S. et al. All-optical histology using ultrashort laser pulses. Neuron 39, 27–41 (2003).

    CAS  PubMed  Google Scholar 

  82. Mainen, Z.F. et al. Two-photon imaging in living brain slices. Methods 18, 231–239, (1999).

    CAS  PubMed  Google Scholar 

  83. Shi, S.H. et al. Rapid spine delivery and redistribution of AMPA receptors after synaptic NMDA receptor activation. Science 284, 1811–1816 (1999).

    CAS  PubMed  Google Scholar 

  84. 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).

    CAS  PubMed  Google Scholar 

  85. Potter, S.M. et al. Structure and emergence of specific olfactory glomeruli in the mouse. J. Neurosci. 21, 9713–9723 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  86. Strome, S. et al. Spindle dynamics and the role of gamma-tubulin in early Caenorhabditis elegans embryos. Mol. Biol. Cell 12, 1751–1764 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  87. 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).

    CAS  PubMed  Google Scholar 

  88. Lawson, N.D. & Weinstein, B.M. In vivo imaging of embryonic vascular development using transgenic zebrafish. Dev. Biol. 248, 307–318 (2002).

    CAS  PubMed  Google Scholar 

  89. Bestvater, F. et al. Two-photon fluorescence absorption and emission spectra of dyes relevant for cell imaging. J. Microsc. 208, 108–115 (2002).

    CAS  PubMed  Google Scholar 

  90. 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).

    PubMed  Google Scholar 

  91. Periasamy, A. Fluorescence resonance energy transfer microscopy: a mini review. J. Biomed. Opt. 6, 287–291 (2001).

    CAS  PubMed  Google Scholar 

  92. 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).

    CAS  PubMed  Google Scholar 

  93. 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).

    CAS  PubMed  Google Scholar 

  94. 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).

    Google Scholar 

  95. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  96. 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).

    CAS  Google Scholar 

  97. 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).

    CAS  PubMed  Google Scholar 

  98. Sheppard, C.J.R. & Gu, M. Image-formation in 2-photon fluorescence microscopy. Optik 86, 104–106 (1990).

    CAS  Google Scholar 

  99. 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).

    Google Scholar 

  100. Sheppard, C.J.R. & Matthews, H.J. Imaging in high-aperture optical systems. J. Opt. Soc. Am. A 4, 1354–1360 (1987).

    Google Scholar 

  101. Beaurepaire, E., Oheim, M. & Mertz, J. Ultra-deep two-photon fluorescence excitation in turbid media. Opt. Commun. 188, 25–29 (2001).

    CAS  Google Scholar 

  102. 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).

    CAS  PubMed  Google Scholar 

  103. 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).

    Google Scholar 

  104. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  105. 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).

    CAS  PubMed  Google Scholar 

  106. Hopkins, J. & Sibbett, W. Ultrashort lasers: big payoff in a flash. Sci. Am. 283, 73–79 (2000).

    Google Scholar 

  107. Soeller, C. & Cannell, M.B. Construction of a two-photon microscope and optimization of illumination pulse duration. Pflugers Arch. 432, 555–561 (1996).

    CAS  PubMed  Google Scholar 

  108. 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).

    CAS  Google Scholar 

  109. 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).

    CAS  PubMed  Google Scholar 

  110. 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).

    CAS  Google Scholar 

  111. 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).

    CAS  PubMed  Google Scholar 

  112. Majewska, A., Yiu, G. & Yuste, R. A custom-made two-photon microscope and deconvolution system. Pflugers Arch. 441, 398–408 (2000).

    CAS  PubMed  Google Scholar 

  113. 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).

    Google Scholar 

  114. 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).

    PubMed  Google Scholar 

  115. Pawley, J.B. Handbook of Biological Confocal Microscopy, edn 2. (Plenum Press, New York, 1995).

    Google Scholar 

  116. Fan, G.Y. et al. Video-rate scanning two-photon excitation fluorescence microscopy and ratio imaging with cameleons. Biophys. J. 76, 2412–2420 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  117. 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).

    CAS  PubMed  Google Scholar 

  118. Gauderon, R., Lukins, P.B. & Sheppard, C.J. Effect of a confocal pinhole in two-photon microscopy. Microsc. Res. Technol. 47, 210–214 (1999).

    CAS  Google Scholar 

  119. 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).

    CAS  PubMed  Google Scholar 

  120. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  121. 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).

    CAS  PubMed  Google Scholar 

  122. 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).

    PubMed  Google Scholar 

  123. Moreaux, L., Sandre, O. & Mertz, J. Membrane imaging by second-harmonic generation microscopy. J. Opt. Soc. Am. B 17, 1685–1694 (2000).

    CAS  Google Scholar 

  124. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  125. 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).

    CAS  PubMed  Google Scholar 

  126. 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).

    CAS  PubMed  Google Scholar 

  127. Mohler, W., Millard, A.C. & Campagnola, P.J. Second harmonic generation imaging of endogenous structural proteins. Methods 29, 97–109 (2003).

    CAS  PubMed  Google Scholar 

  128. 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).

    CAS  PubMed  Google Scholar 

  129. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  130. Hopt, A. & Neher, E. Highly nonlinear photodamage in two-photon fluorescence microscopy. Biophys. J. 80, 2029–2036 (2001).

    CAS  PubMed  PubMed Central  Google Scholar 

  131. 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).

    CAS  Google Scholar 

  132. Patterson, G.H. & Piston, D.W. Photobleaching in two-photon excitation microscopy. Biophys. J. 78, 2159–2162 (2000).

    CAS  PubMed  PubMed Central  Google Scholar 

  133. Neil, M.A. et al. Adaptive aberration correction in a two-photon microscope. J. Microsc. 200 (Pt 2), 105–108 (2000).

    PubMed  Google Scholar 

  134. 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).

    PubMed  Google Scholar 

  135. Marsh, P.N., Burns, D. & Girkin, J.M. Practical implementation of adaptive optics in multiphoton microscopy. Opt. Express 11, 1123–1130 (2003).

    CAS  PubMed  Google Scholar 

  136. 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).

    CAS  PubMed  Google Scholar 

  137. 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).

    CAS  PubMed  Google Scholar 

  138. Jung, J.C. & Schnitzer, M.J. Multiphoton endoscopy. Opt. Lett. 28, 902–904 (2003).

    PubMed  Google Scholar 

  139. Bird, D. & Gu, M. Two-photon fluorescence endoscopy with a mirco-optic scanning head. Opt. Lett. 28, 1552–1554 (2003).

    PubMed  Google Scholar 

  140. Ouzounov, D.G. et al. Delivery of nanojoule femtosecond pulses through large-core microstructured fibers. Opt. Lett. 27, 1513–1515 (2002).

    CAS  PubMed  Google Scholar 

  141. 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).

    PubMed  Google Scholar 

  142. 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).

    CAS  PubMed  Google Scholar 

  143. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

  144. 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).

    CAS  PubMed  PubMed Central  Google Scholar 

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  1. School of Applied and Engineering Physics, 212 Clark Hall, Cornell University, Ithaca, 14853, New York, USA

    Warren R Zipfel, Rebecca M Williams & Watt W Webb

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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

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