UNIT 4.11 Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues

  1. Richard K.P. Benninger1,
  2. David W. Piston2

Published Online: 1 JUN 2013

DOI: 10.1002/0471143030.cb0411s59

Current Protocols in Cell Biology

Current Protocols in Cell Biology

How to Cite

Benninger, R. K. and Piston, D. W. 2013. Two-Photon Excitation Microscopy for the Study of Living Cells and Tissues. Current Protocols in Cell Biology. 59:4.11:4.11.1–4.11.24.

Author Information

  1. 1

    University of Colorado, Anschutz Medical Campus, Aurora, Colorado

  2. 2

    Vanderbilt University Medical Center, Nashville, Tennessee

Publication History

  1. Published Online: 1 JUN 2013
  2. Published Print: JUN 2013


Two-photon excitation microscopy is an alternative to confocal microscopy that provides advantages for three-dimensional and deep tissue imaging. This unit will describe the basic physical principles behind two-photon excitation and discuss the advantages and limitations of its use in laser-scanning microscopy. The principal advantages of two-photon microscopy are reduced phototoxicity, increased imaging depth, and the ability to initiate highly localized photochemistry in thick samples. Practical considerations for the application of two-photon microscopy will then be discussed, including recent technological advances. This unit will conclude with some recent applications of two-photon microscopy that highlight the key advantages over confocal microscopy and the types of experiments which would benefit most from its application. Curr. Protoc. Cell Biol. 59:4.11.1-4.11.24. © 2013 by John Wiley & Sons, Inc.


  • fluorescence;
  • microscopy;
  • two-photon excitation;
  • confocal microscopy