Phase‐resolved fluorescence lifetime measurements for flow cytometry

Pinsky, Bertram G.; Ladasky, John J.; Lakowicz, Joseph R.; Berndt, Klaus W.; Hoffman, R. A.

doi:10.1002/cyto.990140204

Cited by 70 publications

(60 citation statements)

References 18 publications

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“…Fluorescence lifetime has been developed and introduced in flow cytometry using different platforms [10][11][12][13], with more recent advancement in digital frequency-domain techniques [14]. Frequency-domain theory involves modulating the laser excitation source at a continuous radio frequency followed by a measurement of fluorescence lifetime by capturing a time-delayed characteristic of the emission signal.…”

Section: Introductionmentioning

confidence: 99%

Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

Gohar

Cao

Jenkins

et al. 2013

Biomed. Opt. Express

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Intracellular protein transport and localization to subcellular regions are processes necessary for normal protein function. Fluorescent proteins can be fused to proteins of interest to track movement and determine localization within a cell. Currently, fluorescence microscopy combined with image processing is most often used to study protein movement and subcellular localization. In this contribution we evaluate a high-throughput time-resolved flow cytometry approach to correlate intracellular localization of human LC3 protein with the fluorescence lifetime of enhanced green fluorescent protein (EGFP). Subcellular LC3 localization to autophagosomes is a marker of the cellular process called autophagy. In breast cancer cells expressing native EGFP and EGFP-LC3 fusion proteins, we measured the fluorescence intensity and lifetime of (i) diffuse EGFP (ii) punctate EGFP-LC3 and (iii) diffuse EGFP-ΔLC3 after amino acid starvation to induce autophagy-dependent LC3 localization. We verify EGFP-LC3 localization with low-throughput confocal microscopy and compare to fluorescence intensity measured by standard flow cytometry. Our results demonstrate that time-resolved flow cytometry can be correlated to subcellular localization of EGFP fusion proteins by measuring changes in fluorescence lifetime.

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Section: Introductionmentioning

confidence: 99%

Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

Gohar

Cao

Jenkins

et al. 2013

Biomed. Opt. Express

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“…Measurement of fluorescence lifetime using phasesensitive electronics has recently provided an additional parameter for flow cytometry analysis (Deka et al 1994;Pinsky et al 1993;Steinkamp and Crissman 1993;). Phase-sensitive flow cytometry provides the capability to quantify fluorescence lifetimes and to resolve the emissions of multiple fluorochrome labels that have different lifetime values (Steinkamp and Crissman 1993).…”

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confidence: 99%

Differential Effects of Deuterium Oxide on the Fluorescence Lifetimes and Intensities of Dyes with Different Modes of Binding to DNA

Sailer

Nastasi

Valdez

et al. 1997

J Histochem Cytochem.

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SUMMARY Deuterium oxide (D 2 O) increases both the fluorescence lifetime and the fluorescence intensity of the intercalating dyes propidium iodide (PI) and ethidium bromide (EB) when bound to nucleic acid structures. We have used spectroscopic analysis coupled with conventional and phase-sensitive flow cytometry to compare the alterations in intensity and lifetime of various DNA-binding fluorochromes bound to DNA and Chinese hamster ovary (CHO) cells in the presence of D 2 O vs phosphate-buffered saline (PBS). Spectroscopic and flow cytometric studies showed a differential enhancement of intensity and lifetime based on the mode of fluorochrome-DNA interaction. The fluorescence properties of intercalating probes, such as 7-aminoactinomycin D (7-AAD) and ethidium homodimer II (EthD II) were enhanced to the greatest degree, followed by the probes TOTO and YOYO, and the non-intercalating probes Hoechst 33342 (HO) and 4 Ј ,6-diamidino-2-phenylindole (DAPI). The non-intercalating probe mithramycin (MI) gave unexpected results, showing a great enhancement of fluorescence intensity and lifetime in D 2 O, indicating that when staining is performed in PBS, much of the MI fluorescence is quenched by the solvent environment. Apoptotic subpopulations of HL-60 cells had a shorter lifetime compared to nonapoptotic subpopulations when stained with EthD II. These results indicate that accessibility of the dye molecules to the solvent environment, once bound to DNA, leads to the differential enhancement effects of D 2 O on fluorescence intensity and lifetime of these probes. D na-binding fluorochromes have been used extensively in many applications, including flow cytometry and gel and capillary electrophoresis. Fluorochromes utilized in these applications must satisfy several criteria. They must bind specifically to nucleic acids, there must be a low quantum yield as free dye in solution, and there must be enhancement of quantum yield on binding to nucleic acid structures. Several classes of DNA-specific fluorochromes with different modes of base pair ( bp ) binding are available: (a) the DNA intercalators propidium iodide (PI) and ethidium bromide (EB), which lack appreciable bp specificity; (b) DNA intercalators with A-T bp preference, such as ethidium homodimer II (EthD II) or with G-C preference, such as 7-amino actinomycin D (7-AAD); and (c) non-intercalating probes with either A-T bp specificity, such as Hoechst 33342 (HO) or 4 Ј ,6-diamidino-2-phenylindole (DAPI) or with G-C specificity, such as mithramycin (MI). Two high quantum yield cyanine fluorochromes, TOTO and YOYO, are dimers of the dyes thiazole orange and oxazole yellow, respectively (Lee et al. 1986). These fluorochromes are believed to bind to DNA by intercalation, without base specificity (Haugland 1992) but with some base sequence preference (Netzel et al. 1995).Enhancement of the fluorescence intensity of DNAbinding probes improves the signal-to-noise ratio, allowing the use of lower dye concentrations and thereby reducing potential self-quenching between d...

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“…Generally these measurements have been performed using conventional or microscope-based fluorimeters with time-resolved ( 5,2 1 ) or frequency-resolved ( 10,17) methods. Adaptation of fluorescence lifetime measurements to flow cytometry (23,24,26) Exponentially growing HL-60 cells stained with PI or EB had a larger fluorescence lifetime than CHO cells, whether stained in PBS or D,O. Apoptotic HL-60 cell subpopulations with altered chromatin structure exhibited fluorescence intensity enhancement in D,O to a different degree than the nonapoptotic portion of the population.…”

mentioning

confidence: 99%

Interactions of intercalating fluorochromes with DNA analyzed by conventional and fluorescence lifetime flow cytometry utilizing deuterium oxide

et al. 1996

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Phase‐resolved fluorescence lifetime measurements for flow cytometry

Cited by 70 publications

References 18 publications

Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

Subcellular localization-dependent changes in EGFP fluorescence lifetime measured by time-resolved flow cytometry

Differential Effects of Deuterium Oxide on the Fluorescence Lifetimes and Intensities of Dyes with Different Modes of Binding to DNA

Interactions of intercalating fluorochromes with DNA analyzed by conventional and fluorescence lifetime flow cytometry utilizing deuterium oxide

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