Multi‐Photon Imaging

Krishnan Padmanabhan1, Shane E. Andrews2, James. A.J. Fitzpatrick3

1 Center for the Neural Basis of Cognition, Carnegie Mellon Institute, Pittsburgh, Pennsylvania, 2 Howard Hughes Medical Institute and Gene Expression Laboratory, Salk Institute for Biological Studies, La Jolla, California, 3 Waitt Advanced Biophotonics Center, Salk Institute for Biological Studies, La Jolla, California
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 2.9
DOI:  10.1002/0471142956.cy0209s54
Online Posting Date:  October, 2010
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Multi‐photon microscopy, now in its twentieth year, has developed into one of the most robust and powerful techniques for live cell and in vivo fluorescence imaging. Although its theoretical framework is nearly a century old, it has only become a practical tool for biological research with the development of ultrafast lasers and scanning microscopy techniques. In this unit, we outline the basic principles of multi‐photon microscopy, paying special attention to technical considerations for biological applications. Furthermore, we discuss some common applications of the technique, mainly in the field of live cell and in vivo imaging. We illustrate how multi‐photon microscopy can be utilized to address questions ranging from structural cell changes to trafficking of membrane proteins in living organisms, often with resolutions of hundreds of milliseconds. We conclude by outlining the necessary elements needed to establish a successful two‐photon microscopy system. Curr. Protoc. Cytom. 54:2.9.1‐2.9.12. © 2010 by John Wiley & Sons, Inc.

Keywords: multi‐photon microscopy; two‐photon microscopy; confocal microscopy; in vivo imaging; live cell biological imaging; laser scanning microscopy

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Table of Contents

  • Introduction
  • Multi‐Photon Microscopy
  • Multi‐Photon Imaging in Practice
  • Concluding Remarks
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
   Adelsberger, H., Garaschuk, O., and Konnerth, A. 2005. Cortical calcium waves in resting newborn mice. Nat. Neurosci. 8:988‐990.
   Albota, M.A., Xu, C., and Webb, W.W., 1998. Two‐photon fluorescence excitation cross sections of biomolecular probes from 690 to 960 nm. Appl. Opt. 37:7352‐7356.
   Bewersdorf, J., Pick, R., and Hell, S.W. 1998. Multifocal multiphoton microscopy. Opt. Lett. 23:655‐657.
   Bianchini, P. and Diaspro, A. 2008. Three‐dimensional (3D) backward and forward second harmonic generation (SHG) microscopy of biological tissues. J. Biophotonics 1:443‐450.
   Bonhoeffer, T. and Yuste, R. 2002. Spine motility. Phenomenology, mechanisms, and function. Neuron 35:1019‐1027.
   Booth, M.J. and Hell, S.W. 1998. Continuous wave excitation two‐photon fluorescence microscopy exemplified with the 647‐nm ArKr laser line. J. Microsc. 190:298‐304.
   Brakenhoff, G.J., van Spronsen, E.A., van der Voort, H.T., and Nanninga, N. 1989. Three‐dimensional confocal fluorescence microscopy. Methods Cell Biol. 30:379‐398.
   Brustein, E., Marandi, N., Kovalchuk, Y, Drapeau, P, and Konnerth, A. 2003. “In vivo” monitoring of neuronal network activity in zebrafish by two‐photon Ca(2+) imaging. Pflugers Arch. 446:766‐773.
   Carriles, R., Schafer, D.N., Sheetz, K.E., Field, J.J., Cisek, R., Barzda, V., Sylvester, A.W., and Squier, J.A. 2009. Invited review article: Imaging techniques for harmonic and multiphoton absorption fluorescence microscopy. Rev. Sci. Instrum. 80:081101.
   Chen, H., Farkas, E.R., and Webb, W.W. 2008. Chapter 1: In vivo applications of fluorescence correlation spectroscopy. Methods Cell Biol. 89:3‐35.
   Curley, P.F., Ferguson, A.I., White, J.G., and Amos, W.B. 1992. Application of a femtosecond self‐sustaining mode‐locked Ti:Sapphire laser to the field of laser scanning confocal microscopy. Opt. Quantum Electr. 24:851‐859.
   Denk, W., Delaney, K.R., Gelperin, A., Kleinfeld, D., Strowbridge, B.W., Tank, D.W., and Yuste, R. 1994. Anatomical and functional imaging of neurons using 2‐photon laser scanning microscopy. J. Neurosci. Methods 54:151‐162.
   Denk, W., Strickler, J.H., and Webb, W.W. 1990. Two‐photon laser scanning fluorescence microscopy. Science 248:73‐76.
   Denk, W., Yuste, R., Svoboda, K., and Tank, D.W. 1996. Imaging calcium dynamics in dendritic spines. Curr. Opin. Neurobiol. 6:372‐378.
   Diaspro, A., Chirico, G., and Collini, M. 2005. Two‐photon fluorescence excitation and related techniques in biological microscopy. Q. Rev. Biophys. 38:97‐166.
   Eilers, J. and Konnerth, A. 2009. Dye loading with patch pipettes. CSH Protoc. 2009:pdb prot5201.
   Feng, G., Mellor, R.H., Bernstein, M., Keller‐Peck, C., Nguyen, Q.T., Wallace, M., Nerbonne, J.M., Lichtman, J.W., and Sanes, J.R. 2000. Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28:41‐51.
   Fork, R.L., Martinez, O.E., and Gordon, J.P. 1984. Negative dispersion using pairs of prisms. Optics Lett. 9:150‐152.
   Garaschuk, O., Milos, R.I., and Konnerth, A. 2006. Targeted bulk‐loading of fluorescent indicators for two‐photon brain imaging in vivo. Nat. Protoc. 1:380‐386.
   Gobel, W., Kampa, B.M., and Helmchen, F. 2007. Imaging cellular network dynamics in three dimensions using fast 3D laser scanning. Nat. Methods 4:73‐79.
   Goppert‐Mayer, M. 1931. Über Elementarakte mit zwei Quantensprungen. Ann. Physik 401:273‐294.
   Gray, N.W., Weimer, R.M., Bureau, I., and Svoboda, K. 2006. Rapid redistribution of synaptic PSD‐95 in the neocortex in vivo. PLoS Biol. 4:e370.
   Grutzendler, J., Kasthuri, N., and Gan, W.B. 2002. Long‐term dendritic spine stability in the adult cortex. Nature 420:812‐816.
   Heim, R., Cubitt, A.B., and Tsien, R.Y. 1995. Improved green fluorescence. Nature 373:663‐664.
   Helmchen, F., Svoboda, K., Denk, W., and Tank, D.W. 1999. In vivo dendritic calcium dynamics in deep‐layer cortical pyramidal neurons. Nat. Neurosci. 2:989‐996.
   Hernandez, F.E., Belfield, K.D., Cohanoschi, I., Balu, M., and Schafer, K.J. 2004. Three‐ and four‐photon absorption of a multiphoton absorbing fluorescent probe. Appl. Optics 43:5394‐5398.
   Huang, S., Heikal, A.A., and Webb, W.W. 2002. Two‐photon fluorescence spectroscopy and microscopy of NAD(P)H and flavoprotein. Biophys. J. 82:2811‐2825.
   Jontes, J.D., Emond, M.R., and Smith, S.J. 2004. In vivo trafficking and targeting of N‐cadherin to nascent presynaptic terminals. J. Neurosci. 24:9027‐9034.
   Majewska, A., Yiu, G., and Yuste, R. 2000. A custom‐made two‐photon microscope and deconvolution system. Pflugers Arch. 441:398‐408.
   McCurry, C.L., Shepherd, J.D., Tropea, D., Wang, K.H., Bear, M.F., and Sur, M. 2010. Loss of Arc renders the visual cortex impervious to the effects of sensory experience or deprivation. Nat. Neurosci. 13:450‐457.
   Meyer, M.P. and Smith, S.J. 2006. Evidence from in vivo imaging that synaptogenesis guides the growth and branching of axonal arbors by two distinct mechanisms. J. Neurosci. 26:3604‐3614.
   Neher, E. 1995. The use of fura‐2 for estimating Ca buffers and Ca fluxes. Neuropharmacology 34:1423‐1442.
   Neher, E. 2001. Calcium signals and synaptic short‐term plasticity in the central nervous system. Ann. R. Acad. Nac. Med. 118:683‐693.
   Niell, C.M., Meyer, M.P., and Smith, S.J. 2004. In vivo imaging of synapse formation on a growing dendritic arbor. Nat. Neurosci. 7:254‐260.
   Ohki, K., Chung, S., Ch'ng, Y.H., Kara, P., and Reid, R.C. 2005. Functional imaging with cellular resolution reveals precise micro‐architecture in visual cortex. Nature 433:597‐603.
   Ohki, K., Chung, S., Kara, P., Hübener, M., Bonhoeffer, T., and Reid, R.C. 2006. Highly ordered arrangement of single neurons in orientation pinwheels. Nature 442:925‐928.
   Patterson, G.H. and Piston, D.W. 2000. Photobleaching in two‐photon excitation microscopy. Biophys. J. 78:2159‐2162.
   Piston, D.W., Masters, B.R., and Webb, W.W. 1995. Three‐dimensionally resolved NAD(P)H cellular metabolic redox imaging of the in situ cornea with two‐photon excitation laser scanning microscopy. J. Microsc. 178:20‐27.
   Portera‐Cailliau, C., Weimer, R.M., De Paola, V., Caroni, P., and Svoboda, K. 2005. Diverse modes of axon elaboration in the developing neocortex. PloS Biol. 3:1473‐1487.
   Stettler, D.D., Yamahachi, H., Li, W., Denk, W., and Gilbert, C.D. 2006. Axons and synaptic boutons are highly dynamic in adult visual cortex. Neuron 49:877‐887.
   Stosiek, C., Garaschuk, O., Holthoff, K., and Konnerth, A. 2003. In vivo two‐photon calcium imaging of neuronal networks. Proc. Natl. Acad. Sci. U.S.A. 100:7319‐7324.
   Trachtenberg, J.T., Chen, B.E., Knott, G.W., Feng, G., Sanes, J.R., Welker, E., and Svoboda, K. 2002. Long‐term in vivo imaging of experience‐dependent synaptic plasticity in adult cortex. Nature 420:788‐794.
   Wang, K.H., Majewska, A., Schummers, J., Farley, B., Hu, C., Sur, M., and Tonegawa, S. 2006. In vivo two‐photon imaging reveals a role of arc in enhancing orientation specificity in visual cortex. Cell 126:389‐402.
   Xu, C., Guild, J., Webb, W., and Denk, W. 1995. Determination of absolute two‐photon excitation cross sections by in situ second‐order autocorrelation. Opt. Lett. 20:2372.
   Xu, C., Zipfel, W., Shear, J.B., Williams, R.M., and Webb, W.W. 1996. Multiphoton fluorescence excitation: New spectral windows for biological nonlinear microscopy. Proc. Natl. Acad. Sci. U.S.A. 93:10763‐10768.
   Yamahachi, H., Marik, S.A., McManus, J.N., Denk, W., and Gilbert, C.D. 2009. Rapid axonal sprouting and pruning accompany functional reorganization in primary visual cortex. Neuron 64:719‐729.
   Yuste, R. and Bonhoeffer, T. 2001. Morphological changes in dendritic spines associated with long‐term synaptic plasticity. Ann. Rev. Neurosci. 24:1071‐1089.
   Yuste, R. and Bonhoeffer, T. 2004. Genesis of dendritic spines: Insights from ultrastructural and imaging studies. Nat. Rev. Neurosci. 5:24‐34.
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