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Microscopy Reference Library

Multiphoton Microscopy

The application of nonlinear excitation techniques to the imaging of synthetic fluorophores and fluorescent proteins in biology and medicine has witnessed increasing attention over the past several years, primarily due to the introduction of turnkey pulsed laser systems coupled to advanced instrumentation. The references described in this section contain review articles and original research reports on multiphoton microscopy with emphasis on the theoretical background, microscope configuration, specimen preparation, deep tissue imaging, and numerous applications.

Piston, D. W.

Imaging living cells and tissues by two-photon excitation microscopyTrends in Cell Biology 9: 66-69 (1999).  An excellent "starter" review article for investigators that are entering the field. The author discusses the principles of multiphoton microscopy, practical considerations in executing the technique, and several examples of the application of this methodology in cell biology.

Diaspro, A., Chirico, G. and Collini, M.

Two-photon fluorescence excitation and related techniques in biological microscopy.  Quarterly Reviews of Biophysics 38: 97-166 (2005).  One of the most comprehensive articles available on multiphoton microscopy. Discussed are the historical background, basic principles, fluorescent probe characteristics, microscope design considerations, and applications. The paper contains hundreds of references for additional information on the technique.

Dunn, K. W. and Young, P. A.

Principles of multiphoton microscopy.  Nephron Experimental Nephrology 103: e33-e40 (2006).  The authors discuss general principles of multiphoton excitation and compare this technique with single-photon laser scanning confocal microscopy. Sections on the utilization of multiphoton fluorescence in biomedical research and the practical aspects of multiphoton imaging are included.

Benninger, R. K. P., Hao, M. and Piston, D. W.

Multi-photon excitation imaging of dynamic processes in living cells and tissues.  Reviews of Physiology, Biochemistry and Pharmacology 160: 71-92 (2008).  The authors describe how turkey ultrafast laser systems are enabling greater access to multiphoton technology in cell biology laboratories. Also discussed are uses of multiphoton microscopy in the neurosciences, developmental biology, immunology, cancer biology, and endocrinology.

Helmchen, F. and Denk, W.

Deep tissue two-photon microscopy.  Nature Methods 2: 932-940 (2005).  A review of imaging deep within tissues and live animals using multiphoton and related nonlinear methodology. The article describes nonlinear effects, light scattering, microscope configuration parameters, image formation, resolution, and numerous other experimental variables.

Oheim, M., Michael, D. J., Geisbauer, M., Madsen, D. and Chow, R. H.

Principles of two-photon excitation fluorescence microscopy and other nonlinear imaging approaches.  Advanced Drug Delivery Reviews 58: 788-808 (2006).  This excellent review covers the basic principles of multiphoton microscopy and compares the advantages and disadvantages to other techniques. Also discussed are the use of nonlinear excitation to generate resolution beyond the diffraction limit and progress in non-scanning technology, which is potentially useful in screen applications.

Svoboda, K. and Yasuda, R.

Principles of two-photon excitation microscopy and its applications to neuroscience.  Neuron 50: 823-839 (2006).  Targeting applications for multiphoton microscopy in the study of brain structure and function, the authors review the basic principles underlying the technique and highlight recent applications. In addition, the limitations for investigations in the neurosciences are discussed, as well as speculation about future research and development. Included is a large collection of pertinent references.

Rubart, M.

Two-photon microscopy of cells and tissues.  Circulation Research 95: 1154-1166 (2004).  The author provides a comprehensive overview of the use of two-photon microscopy in selected applications to assess cellular and subcellular events in intact, strongly scattering tissue. Included are discussions of the imaging calcium dynamics in living cardiomyocytes and the potential applications of multiphoton microscopy to integrative cardiac physiology.

Hänninen, P., Soukka, J. and Soini, J. T.

Two-photon excitation fluorescence bioassays.  Annals of the New York Academy of Sciences 1130: 320-326 (2008).  This review focuses on the application of multiphoton microscopy to non-imaging bioassay techniques. Included are discussions of diagnostic and research assays, including instrumentation, optimizing assay performance, and limitations of the technology.

Denk, W., Strickler, J. H. and Webb, W. W.

Two-photon laser scanning fluorescence microscopy.  Science 248: 73-76 (1990).  The original research report on two-photon excitation for imaging with laser scanning microscopy. The authors describe the technique and provide several examples of biological applications using synthetic fluorophores.