Quantitative microfluorometry of isolated living cells with pulsed excitation: Development of an effective and relatively inexpensive instrument

Abstract

Using a computer interfaced original apparatus, we can analyze the fluorescence decay time of a single living cell. This apparatus can measure rate constants in the range of one to few hundred nanoseconds. The smallest sample analyzed was 5×5×5 μm. We obtained enough sensitivity to measure the fluorescence of about 105 molecules of benzo(a)pyrene with a signal/noise ratio equal to 5. The signal was then defined with 300 points and experiment was 60 s long with a laser pulse frequency equal to 100 Hz. Distortions were less than 3% of the signal. Different kinds of information became accessible: statistical comparison on cellular populations and variation with time of studied parameters. As an example of applications, the intrinsic cell fluorescence has been analyzed in order to determine the amounts of bound and free NAD(P)H in single living cells.