Nanosecond fluorescence microscopy. Emission kinetics of fura-2 in single cells

A microscope based time-correlated single photon counting instrument has been constructed to measure fluorescence intensity and emission anisotropy decays from fluorophores in single cells on a nanosecond time scale. The sample is excited and the emission collected using epi-illumination optics with...

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Bibliographic Details
Published in:Biophysical journal 1991, Vol.59 (1), p.186-202
Main Authors: Keating, S.M., Wensel, T.G.
Format: Article
Language:English
Subjects:
Animals
Biological and medical sciences
Cell Line
Cell Physiological Phenomena
Fundamental and applied biological sciences. Psychology
Fura-2
General aspects, investigation technics, apparatus
Indicators and Reagents
Kinetics
Leukemia, Basophilic, Acute
Leukemia, Experimental
Mathematics
Microscopy, Fluorescence - methods
Models, Theoretical
Rats
Time Factors
Tissues, organs and organisms biophysics
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Summary:A microscope based time-correlated single photon counting instrument has been constructed to measure fluorescence intensity and emission anisotropy decays from fluorophores in single cells on a nanosecond time scale. The sample is excited and the emission collected using epi-illumination optics with frequency-doubled pulses from the cavity-dumped output of a synchronously pumped dye laser serving as an excitation source. Collection of decays from a single cell is possible due to the presence of an iris in the emission path that can be reduced to less than the diameter of a single cell. Using the instrument the decay of 60 nM 1,6-diphenyl-1,3,5-hexatriene was measured, demonstrating that adequate data for lifetime analysis can be recorded from fewer 10(3) molecules of the fluorophore in an illuminated volume of 23 fl. In addition, the intensity and anisotropy decays of fura-2 in single adherent cells and in suspensions of fura-2 loaded cells in suspension, although the relative amplitudes and decay constants vary somewhat from cell to cell. The results indicate that a significant but variable fraction of fura-2 is bound to relatively immobile macromolecular components in these cells.
ISSN:0006-3495
1542-0086
DOI:10.1016/S0006-3495(91)82210-X