Time‐Resolved Fluorescence Measurements

John A. Steinkamp1

1 Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 1.15
DOI:  10.1002/0471142956.cy0115s11
Online Posting Date:  May, 2001
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This unit provides a detailed review and technical discussion of the principles of time‐resolved fluorescence. The unique feature of this particular unit is that the principles are directly tied to the use of flow cytometry. There are a number of advantages in being able to discriminate between fluorochromes based upon their lifetimes as opposed to their emission wavelengths. One particularly good application is the determination of bound versus free molecules. The author covers both the theoretical and practical components of measuring fluorescence in the time domain. In addition, the unit provides a listing of the most common fluorescent dyes with comparative data of lifetime and excitation conditions.

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Table of Contents

  • Excited‐State Lifetime Measurements
  • Theory of Frequency‐Domain (Single‐Frequency) Lifetime Measurements Applied to Flow Cytometry
  • FLFCM Instrumentation and Signal Processing
  • Recent Applications of FLFCM
  • Summary
  • Literature Cited
  • Figures
  • Tables
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Literature Cited

Literature Cited
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  This work was performed at the Los Alamos National Laboratory under the joint sponsorship of the United States Department of Energy and the Los Alamos National Flow Cytometry Resource (National Institutes of Health Grants P41‐RR013150 and R01‐RR07855). The author wishes to thank Nancy M. Lehnert and Harry A. Crissman for their assistance in cell preparation and staining.
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