Fluorescence intensity and lifetime measurement of free and particle-bound fluorophore in a sample stream by phase-sensitive flow cytometry

We report a novel method to quantify fluorescence intensity and lifetime of free (solution) and particle-bound fluorophore in a flow stream. The technique combines flow cytometry and frequency-domain lifetime spectroscopy principles to make unique fluorescence measurements on free fluorophore and fluorophore-labeled particles. Fluorophore-labeled microspheres suspended in a fluorophore solution are analyzed as they flow through a chamber and pass across an intensity-modulated laser beam consisting of a continuous-wave (cw) direct-current (dc) and high-frequency (sine wave) excitation component. Fluorescence emission signals consisting of a dc-offset steady-state sinusoidal signal (fluorophore solution) and a sinusoidally modulated Gaussian-shaped signal pulse (fluorophore-labeled particles) are processed electronically to quantify intensities and lifetimes. The cw-excited, particle-associated fluorophore (pulse) and steady-state (dc) solution fluorescence intensity signals are measured using low-pass filt...

[1]  J. Lakowicz,et al.  Fluorescence detection of the anticancer drug topotecan in plasma and whole blood by two-photon excitation. , 1996, Analytical biochemistry.

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

[3]  J. Steinkamp,et al.  Monitoring uptake of ellipticine and its fluorescence lifetime in relation to the cell cycle phase by flow cytometry. , 1997, Experimental Cell Research.

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

[5]  P. So,et al.  Chapter 14 Fluorescence Lifetime Imaging Techniques for Microscopy , 1998 .

[6]  Joseph R. Lakowicz,et al.  A 10‐GHz frequency‐domain fluorometer , 1990 .

[7]  J. Steinkamp,et al.  Analysis of fluorescence lifetime and quenching of FITC-conjugated antibodies on cells by phase-sensitive flow cytometry. , 1996, Cytometry.

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

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

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

[11]  D. O'connor,et al.  Time-Correlated Single Photon Counting , 1984 .

[12]  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.

[13]  J A Steinkamp,et al.  Interactions of intercalating fluorochromes with DNA analyzed by conventional and fluorescence lifetime flow cytometry utilizing deuterium oxide. , 1996, Cytometry.

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

[15]  G C Salzman,et al.  Modular electronics for flow cytometry and sorting: the LACEL system. , 1981, Cytometry.

[16]  Enrico Gratton,et al.  Frequency domain time-resolved microscope using a fast-scan CCD camera , 1994, Photonics West - Lasers and Applications in Science and Engineering.

[17]  John A. Steinkamp,et al.  Fluorescence lifetime as a new parameter in analytical cytology measurements , 1996, Photonics West.

[18]  J A Steinkamp,et al.  Fluorescence lifetime measurements in a flow cytometer by amplitude demodulation using digital data acquisition technique. , 1994, Cytometry.

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

[20]  J A Steinkamp,et al.  Differential Effects of Deuterium Oxide on the Fluorescence Lifetimes and Intensities of Dyes with Different Modes of Binding to DNA , 1997, The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society.

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

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

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

[24]  J A Steinkamp,et al.  Flow cytometric fluorescence lifetime analysis of DNA-binding probes. , 1998, European journal of histochemistry : EJH.

[25]  L. B. Robinson,et al.  Reduction of Baseline Shift in Pulse-Amplitude Measurements , 1961 .

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

[27]  J. Steinkamp,et al.  Discrimination of damaged/dead cells by propidium iodide uptake in immunofluorescently labeled populations analyzed by phase-sensitive flow cytometry. , 1999, Journal of immunological methods.

[28]  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 .

[29]  J. Steinkamp,et al.  Simultaneous analysis of relative DNA and glutathione content in viable cells by phase-resolved flow cytometry. , 1999, Cytometry.

[30]  J. Lakowicz.  Principles of fluorescence spectroscopy , 1983 .

[31]  J A Steinkamp,et al.  Resolution of fluorescence signals from cells labeled with fluorochromes having different lifetimes by phase-sensitive flow cytometry. , 1993, Cytometry.

[32]  P H Sydenham,et al.  Phase sensitive detection as a means to recover signals buried in noise , 1975 .

[33]  J A Steinkamp,et al.  Time-resolved fluorescence-decay measurement and analysis on single cells by flow cytometry. , 1996, Applied optics.

[34]  A Kusumi,et al.  Fluorescence lifetime imaging microscopy (flimscopy). Methodology development and application to studies of endosome fusion in single cells. , 1993, Biophysical journal.

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

[36]  J A Steinkamp,et al.  Apoptosis induced with different cycle-perturbing agents produces differential changes in the fluorescence lifetime of DNA-bound ethidium bromide. , 1998, Cytometry.

[37]  M. L. Meade,et al.  Advances in lock-in amplifiers , 1982 .