Capture of Fluorescence Decay Times by Flow Cytometry

Abstract

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.

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

“…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.…”

“…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.…”

Fluorescence lifetime shifts of NAD(P)H during apoptosis measured by time‐resolved flow cytometry

“…A FLFC system was optimized (58)(59)(60)66) and used to demonstrate how screening of individual cells for the autofluorescence lifetime shifts of NAD(P)H is a possible method of counting, and eventually sorting, cells based on metabolic shifts that occur during cell death. Our overall results include an increase in both the autofluorescence intensity and lifetime when apoptosis is induced by using STS.…”

FLIM-FRET for Cancer Applications

“…Cy7 (7 methine protons), a cyanine family member with longer polymethine bridge emits in NIR range but has low quantum yield. Indocyanine green (ICG), belonging to the same family, is the only FDA approved long wavelength NIR fluorescent dye for direct human administration in medical diagnostics such as retinal angiography and hepatic functions [70, 71]. ICG has been used to image tumors and shown to accumulate in tumors, due to the enhanced permeability and retention effect (EPR) [72].…”