Fluorescence lifetime as a new parameter in analytical cytology measurements

A phase-sensitive flow cytometer has been developed to quantify fluorescence decay lifetimes on fluorochrome-labeled cells/particles. This instrument combines flow cytometry (FCM) and frequency-domain fluorescence spectroscopy measurement principles to provide unique capabilities for making phase-resolved lifetime measurements, while preserving conventional FCM capabilities. Cells are analyzed as they intersect a high-frequency, intensity-modulated (sine wave) laser excitation beam. Fluorescence signals are processed by conventional and phase-sensitive signal detection electronics and displayed as frequency distribution histograms. In this study we describe results of fluorescence intensity and lifetime measurements on fluorescently labeled particles, cells, and chromosomes. Examples of measurements on intrinsic cellular autofluorescence, cells labeled with immunofluorescence markers for cell-surface antigens, mitochondria stains, and on cellular DNA and protein binding fluorochromes will be presented to illustrate unique differences in measured lifetimes and changes caused by fluorescence quenching. This innovative technology will be used to probe fluorochrome/molecular interactions in the microenvironment of cells/chromosomes as a new parameter and thus expand the researchers' understanding of biochemical processes and structural features at the cellular and molecular level.

[1]  J. Lakowicz,et al.  Analysis of fluorescence decay kinetics from variable-frequency phase shift and modulation data. , 1984, Biophysical journal.

[2]  Joseph R. Lakowicz,et al.  Phase-modulation fluorometry using a frequency-doubled pulsed laser diode light source , 1990 .

[3]  Jean Vigo,et al.  Quantitative microfluorometry of isolated living cells with pulsed excitation: Development of an effective and relatively inexpensive instrument , 1987 .

[4]  R. D. Spencer,et al.  MEASUREMENTS OF SUBNANOSECOND FLUORESCENCE LIFETIMES WITH A CROSS‐CORRELATION PHASE FLUOROMETER * , 1969 .

[5]  Joseph R Lakowicz,et al.  Phase Fluorometry Using a Continuously Modulated Laser Diode. , 1992, Analytical chemistry.

[6]  Robert M. Clegg,et al.  Fluorescence lifetime imaging microscopy (FLIM): Spatial resolution of microstructures on the nanosecond time scale , 1993 .

[7]  J. Lakowicz,et al.  Phase-resolved fluorescence lifetime measurements for flow cytometry. , 1993, Cytometry.

[8]  E. Gratton,et al.  A continuously variable frequency cross-correlation phase fluorometer with picosecond resolution. , 1983, Biophysical journal.

[9]  Enrico Gratton,et al.  Multifrequency cross‐correlation phase fluorometer using synchrotron radiation , 1984 .

[10]  B. Maliwal,et al.  Construction and performance of a variable-frequency phase-modulation fluorometer. , 1985, Biophysical chemistry.

[11]  J. Lakowicz,et al.  Fluorescence lifetime imaging of free and protein-bound NADH. , 1992, Proceedings of the National Academy of Sciences of the United States of America.

[12]  E. Gratton,et al.  The Measurement and Analysis of Heterogeneous Emissions by Multifrequency Phase and Modulation Fluorometry , 1984 .

[13]  T. Wensel,et al.  Nanosecond fluorescence microscopy. Emission kinetics of fura-2 in single cells. , 1991, Biophysical journal.

[14]  Kerry M. Swift,et al.  48000 MHFTM: a dual-domain Fourier transform fluorescence lifetime spectrofluorometer , 1990, Photonics West - Lasers and Applications in Science and Engineering.

[15]  G. G. Stokes.  "J." , 1890, The New Yale Book of Quotations.

[16]  Richard A. Keller,et al.  Detection and lifetime measurement of single molecules in flowing sample streams by laser‐induced fluorescence , 1993 .

[17]  John A. Steinkamp,et al.  Flow cytometer for resolving signals from heterogeneous fluorescence emissions and quantifying lifetime in fluorochrome-labeled cells/particles by phase-sensitive detection , 1993 .

[18]  James N. Demas,et al.  Excited State Lifetime Measurements , 1983 .

[19]  L. S. Cram,et al.  Simultaneous dual-frequency phase-sensitive flow cytometric measurements for rapid identification of heterogeneous fluorescence decays in fluorochrome-labeled cells and particles. , 1995, Cytometry.

[20]  Joseph R. Lakowicz,et al.  2-GHz frequency-domain fluorometer , 1986 .