A Review of New High-Throughput Methods Designed for Fluorescence Lifetime Sensing From Cells and Tissues

Abstract

Though much of the interest in fluorescence in the past has been on measuring spectral qualities such as wavelength and intensity, there are two other highly useful intrinsic properties of fluorescence: lifetime (or decay) and anisotropy (or polarization). Each has its own set of unique advantages, limitations, and challenges in detection when it comes to use in biological studies. This review will focus on the property of fluorescence lifetime, providing a brief background on instrumentation and theory, and examine the recent advancements and applications of measuring lifetime in the fields of high-throughput fluorescence lifetime imaging microscopy (HT-FLIM) and time-resolved flow cytometry (TRFC). In addition, the crossover of these two methods and their outlooks will be discussed.

Optimization of Advanced Live-Cell Imaging through Red/Near-Infrared Dye Labeling and Fluorescence Lifetime-Based Strategies

“…In our study, experiments were performed through HyD-S, HyD-X and HyD-R that enabled detection of fluorescence signals up to 850 nm. HyD-S is based on silicon multi-pixel photon counter technology, in which incident photons are spread on an array of independent avalanche photodiodes [ 19 , 34 ]. HyD-S had a rather good sensitivity all over the spectrum in this study.…”

“…HyD-S had a rather good sensitivity all over the spectrum in this study. HyD-X and HyD-R are based on hybrid detector technology with a gallium arsenide phosphide photocathode coupled to an avalanche diode [ 19 , 35 ]. HyD-X had better sensitivity in the red spectrum in this study.…”

“…With spectrally close fluorochromes or with close lifetimes, HyD-X was the sole detector used due to its characteristics and its calibration with the FLIM module. Historically, FLIM data were processed by fitting the decay of each pixel using one or several exponentials, leading to identification of the decay times and amplitudes of components [ 16 , 17 , 19 ]. This approach is not simple due to the complexity of decay patterns and information extraction.…”

“…The fluorescence lifetime of small organic fluorophores varies from 0.1 to 20 ns when measured through Time Correlated Single Photon Counting (TCSPC) [ 15 ]. TCSPC packages are now available through user-friendly solutions, leading to democratization of Fluorescence Lifetime Imaging Microscopy (FLIM) [ 18 , 19 ]. FLIM is therefore a potential technique for multiplexing and monitoring the molecular environment of fluorophores [ 20 ].…”

Lipid Membrane Alterations in Tumor Spheroids Revealed by Fluorescence Lifetime Microscopy Imaging

“…17 The fluorescence lifetime is defined as the average time for a fluorescent molecule to return to its ground state from its original excited state. 18 Fluorescence lifetime is an intrinsic parameter of the fluorophore and remains constant under any optical conditions. 19 Timecorrelated single photon counting (TCSPC) is the most commonly used acquisition method for fluorescence lifetimes.…”