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

Steinkamp, John A.; Keij, Jan F.

doi:10.1063/1.1150143

Cited by 18 publications

(27 citation statements)

References 38 publications

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“…Most lifetime work has been investigated with fluorescence lifetime imaging microscopy (FLIM) platforms, and although these measurements were conducted in a well-characterized lifetime cytometer [11][12][13][14][39][40][41] FLIM measurements are indeed helpful to evaluate lifetime gradients across a cell and pixel-by-pixel. Yet flow cytometry has several advantages in that it can help hunt for rare events and reveal trends in population owing to dynamic changes with living cells.…”

Section: Discussionmentioning

confidence: 99%

“…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%

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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: Discussionmentioning

confidence: 99%

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

View full text Add to dashboard Cite

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“…Both approaches have been demonstrated in cytometry with various cell and bead-based applications (Cui et al, 2003, Steinkamp and Keij, 1999, Steinkamp et al, 1993, Deka and Steinkamp, 1996, Jin et al 2007 and 2009, and Steinkamp 2001). With time-domain approaches, the fluorescence decay is observed over time with single photon counting detectors, then measured and fit using decay functions.…”

Section: Introductionmentioning

confidence: 99%

Capture of Fluorescence Decay Times by Flow Cytometry

et al. 2012

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In flow cytometry, the fluorescence decay time of an excitable species has been largely underutilized and is not likely found as a standard parameter on any imaging cytometer, sorting, or analyzing system. Most cytometers lack fluorescence lifetime hardware mainly owing to two central issues. Foremost, research and development with lifetime techniques has lacked proper exploitation of modern laser systems, data acquisition boards, and signal processing techniques. Secondly, a lack of enthusiasm for fluorescence lifetime applications in cells and with bead-based assays has persisted among the greater cytometry community. In this unit, we describe new approaches that address these issues and demonstrate the simplicity of digitally acquiring fluorescence relaxation rates in flow. The unit is divided into protocol and commentary sections in order to provide a most comprehensive discourse on acquiring the fluorescence lifetime with frequency-domain methods. The unit covers (i) standard fluorescence lifetime acquisition (protocol-based) with frequency-modulated laser excitation, (ii) digital frequency-domain cytometry analyses, and (iii) interfacing fluorescence lifetime measurements onto sorting systems. Within the unit is also a discussion on how digital methods are used for aliasing in order to harness higher frequency ranges. Also, a final discussion is provided on heterodyning and processing of waveforms for multi-exponential decay extraction.

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“…Steinkamp et al . were early pioneers of robust PSFC systems that provided reproducible lifetime measurements with sub-nanosecond resolution (3,11). PSFC uses a frequency-domain method in which the cytometer laser excitation source is intensity-modulated at a single radiofrequency (RF).…”

Section: Introductionmentioning

confidence: 99%

Digital analysis and sorting of fluorescence lifetime by flow cytometry

2010

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Frequency-domain flow cytometry techniques are combined with modifications to the digital signal-processing capabilities of the open reconfigurable cytometric acquisition system (ORCAS) to analyze fluorescence decay lifetimes and control sorting. Real-time fluorescence lifetime analysis is accomplished by rapidly digitizing correlated, radiofrequency (RF)-modulated detector signals, implementing Fourier analysis programming with ORCAS' digital signal processor (DSP) and converting the processed data into standard cytometric list mode data. To systematically test the capabilities of the ORCAS 50 MS/sec analog-to-digital converter (ADC) and our DSP programming, an error analysis was performed using simulated light scatter and fluorescence waveforms (0.5-25 ns simulated lifetime), pulse widths ranging from 2 to 15 ls, and modulation frequencies from 2.5 to 16.667 MHz. The standard deviations of digitally acquired lifetime values ranged from 0.112 to [2 ns, corresponding to errors in actual phase shifts from 0.01428 to 1.68. The lowest coefficients of variation (\1%) were found for 10-MHz modulated waveforms having pulse widths of 6 ls and simulated lifetimes of 4 ns. Direct comparison of the digital analysis system to a previous analog phase-sensitive flow cytometer demonstrated similar precision and accuracy on measurements of a range of fluorescent microspheres, unstained cells, and cells stained with three common fluorophores. Sorting based on fluorescence lifetime was accomplished by adding analog outputs to ORCAS and interfacing with a commercial cell sorter with a RF-modulated solid-state laser. Two populations of fluorescent microspheres with overlapping fluorescence intensities but different lifetimes (2 and 7 ns) were separated to $98% purity. Overall, the digital signal acquisition and processing methods we introduce present a simple yet robust approach to phase-sensitive measurements in flow cytometry. The ability to simply and inexpensively implement this system on a commercial flow sorter will allow both better dissemination of this technology and better exploitation of the traditionally underutilized parameter of fluorescence lifetime. Published 2010 Wiley-Liss, Inc. y Key terms fluorescence lifetime; digital flow cytometry; phase-sensitive flow cytometry TIME-resolved methods in flow cytometry were introduced almost two decades ago, and upon their inception, high-throughput measurements of fluorescence decay kinetic parameters were established (1-3). In particular, the average fluorescence lifetime, measured on an event-by-event basis, was used as an independent parameter in cell cycle analysis, quenching studies, free-dye experiments, and other assays to discriminate between fluorophores with closely overlapping emission spectra (3-6). Although a powerful indicator of details affecting fluorescence, the excited state lifetime is largely underutilized by cytometrists. The average fluorescence lifetime can provide a quantitative measure of fluorophore environment yet is not measurable by comme...

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

Cited by 18 publications

References 38 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

Capture of Fluorescence Decay Times by Flow Cytometry

Digital analysis and sorting of fluorescence lifetime by flow cytometry

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