Spectrally resolved fluorescence lifetime imaging: new developments and applications

Abstract

The fluorescence lifetime of different molecular species is calculated from the measured fluorescence intensity decrease following short pulsed laser excitation, by a multi-channel fitting procedure. In a FRET (Förster Resonant Energy Transfer) experiment the time dependent behaviour of the donor profile is assumed in a first view mono-exponential and the acceptor decay profile is solved analytically. A global minimization fitting algorithm has increased information content than a single channel fitting routine. In a normal FRET-FLIM experiment, the efficiency of FRET is calculated only by considering the kinetics of the donor. However, as will be shown, a considerable improvement could be achieved when time-resolved and spectral-resolved techniques are simultaneously incorporated.

A hybrid FLIM-elastic net platform for label free profiling of breast cancer

“…Fluorescence Lifetime Imaging Microscopy (FLIM) is a robust technique 19 with several key advantages: (i) the lifetime of a uorophore is independent of uorophore concentration, photobleaching, initial perturbation conditions, excitation wavelength, light exposure conditions, method of measurement, and laser power, (ii) uorescence lifetime can be sensitive to a variety of internal factors dened by the structure of the uorophore and external factors that include temperature, polarity, and the presence of uorescence quenchers, 20 and (iii) distinct uorescence lifetime in the same pixel originating from uorophores that are excited at the same excitation wavelength can be resolved with appropriate tting functions. 21,22 These characteristics render FLIM as an attractive tool to report on localization, microenvironment, and binding kinetics 23 in a label free manner or using simple reporters. Real-time spatiotemporal alterations of metabolites with innate uorescence might have the ability to provide information on the biochemical/physiological changes within a cell.…”