Measurement of fluorescence decay time in living cells

This paper reports a simple technique for measuring fluorescence decay times of intracellular compounds. The data can be obtained from suspensions of living cells or, using a microscope, from single cells. The method employs a TRW, Inc. nanosecond spectral source and decay time computer which have been used previously for measuring decay times of aqueous solutions. Ascites tumor cells, liver cells, fibroblasts, bacteria, and cell fractions, after incubation with a fluorochrome and appropriate washing, can be suspended in a cuvette (or in the case of single cells, placed on a microscope slide) and the fluorescent decay time can be read out digitally in nanoseconds. The instrument is most accurate where actual decay values are > 2 ns, and under these conditions reads to a precision of ±0.5 ns in clear solutions, and ±1.0 ns for intracellular work. Results obtained using the fluorescent probes, anilinonaphthalene sulfonate (ANS), toluidinyl naphthalene sulfonate (TNS), 3,4-benzpyrene (BP), and 2-aminonaphthalene (2-AN) are presented as examples.