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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Abstract

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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Materials

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Figures

  •   FigureFigure 2.9.1 (A) Schematic energy level diagram illustrating the process of one‐ and two‐photon excitation. Either a single blue photon or two infrared photons connect the ground (S0) and excited (S1) electronic states. (B) Schematic illustrating how fluorescence is only excited at the plane of focus in the two‐photon case, as opposed to throughout the focusing cone in the single photon case. (C) Real photograph of one‐ and two‐photon excitation (Courtesy of Brad Amos, MRC, Cambridge, England)
  •   FigureFigure 2.9.2 Tuning range across the near infrared region of the spectrum of a Ti:Sapphire laser system (red), overlayed with the absorption spectrum of water (black). The green vertical bar illustrates the typical range of two‐photon excitation wavelengths for eGFP.
  •   FigureFigure 2.9.3 (A) Simplified schematic illustration of the optical layout of a typical two‐photon microscope system, comprising a Ti:Sapphire laser, acousto‐optical modulator (AOM) for controlling power output, scanning system, microscope objective, and photomultiplier tube (PMT) for fluorescence detection. (B) Schematic illustrations of the various fluorescence detection geometries in confocal (descanned) and two‐photon (non‐descanned and transmitted) microscopies.
  •   FigureFigure 2.9.4 Image of HB9 GFP‐positive motor neurons from a live neonatal spinal cord. (A) Confocal slice excited at 488 nm and imaged with descanned detection at a depth of 100 µm. Top arrow indicates HB9 GFP‐positive motor neuron cell bodies in the first motor pool. Bottom arrow indicates the expected location of those cell bodies projections. (B) Two‐photon slice of the exact same region reveals much clearer cell bodies (top arrow) and greatly increased resolution of the genetic label in the faint neuronal projections (bottom arrow).

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Literature Cited

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