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

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.

[1]  B. Thorell,et al.  MICROSPECTROFLUOROMETRIC APPROACH TO THE STUDY OF FREE/BOUND NAD(P)H RATIO AS METABOLIC INDICATOR IN VARIOUS CELL TYPES , 1982, Photochemistry and photobiology.

[2]  J. Brochon,et al.  Pulse fluorimetry study of beef liver glutamate dehydrogenase reduced nicotinamide adenine dinucleotide phosphate complexes. , 1976, Biochemistry.

[3]  J. B. Birks,et al.  Photophysics of aromatic molecules , 1970 .

[4]  L. Brand,et al.  Fluorescence decay studies of reduced nicotinamide adenine dinucleotide in solution and bound to liver alcohol dehydrogenase. , 1976, Biochemistry.

[5]  E. Kohen,et al.  32-Msec scan of the NAD(P)H fluorescence spectrum in single living cells. , 1973, The Review of scientific instruments.

[6]  T. I. Quickenden,et al.  Criterion of goodness of fit for deconvolution calculations , 1981 .

[7]  Bernard Valeur,et al.  Analyse des courbes de décroissance multiexponentielles par la méthode des fonctions modulatrices-application à la fluorescence , 1973 .

[8]  Y. Mauss,et al.  Quantum yields and fluorescence lifetimes of acridine derivatives interacting with DNA , 1977, Biopolymers.

[9]  Sidney Udenfriend,et al.  Fluorescence assay in biology and medicine , 1962 .

[10]  P. Wahl,et al.  Mesures des déclins de l'anisotropie de fluorescence de la γ‐globuline et de ses fragments Fab, Fc et F(ab)2 marqués avec le l‐sulfonyl‐5‐diméthyl‐aminonaphtalène , 1972 .

[11]  B. Thorell,et al.  Studies on metabolic events in localized compartments of the living cell by rapid microspectrofluorometry. , 1974, Advances in biological and medical physics.

[12]  T. G. Scott,et al.  Synthetic spectroscopic models related to coenzymes and base pairs. V. Emission properties of NADH. Studies of fluorescence lifetimes and quantum efficiencies of NADH, AcPyADH, [reduced acetylpyridineadenine dinucleotide] and simplified synthetic models , 1970 .

[13]  B. Valeur.  Analysis of time-dependent fluorescence experiments by the method of modulating functions with special attention to pulse fluorometry , 1978 .

[14]  B. Thorell,et al.  Rapid microspectrofluorimetry for biochemical and metabolic studies in single living cells. , 1974, Biochimica et biophysica acta.

[15]  M. Ameloot,et al.  Criteria for model evaluation in the case of deconvolution calculations , 1982 .

[16]  O. Svelto,et al.  FLUORESCENCE OF COMPLEXES OF QUINACRINE MUSTARD WITH DNA. I. INFLUENCE OF THE DNA BASE COMPOSITION ON THE DECAY TIME IN BACTERIA , 1979 .

[17]  Andreas A. Thaer,et al.  Fluorescence Techniques in Cell Biology , 1973, Springer Berlin Heidelberg.

[18]  Preliminary Trial with a Continuous Helium-cadmium Laser as Excitation Source for the Microspectrofluorometric Assay of NAD(P)H in Single Living Cells , 1974 .