Rapid acquisition, analysis, and display of fluorescence lifetime-resolved images for real-time applications

Abstract

Fluorescence lifetime-resolved imaging (FLI) is a relatively new technique of fluorescence imaging whereby the spatial distribution of fluorescence decay times can be determined directly at every pixel of an image simultaneously. The fluorescence decay times of many chromophores can act as sensitive gauges of their molecular environments. By employing measurement techniques that are quantitatively related to the radiative dynamics of the dye molecules (in the nanosecond time range), additional physical parameters are available for discerning different fluorophores with disparate lifetimes, or for characterizing a single fluorophore in different surroundings. Many physical processes such as molecular aggregation, binding of dyes to macromolecular species, inclusion of chromophores in specific cellular organwelles, fluorescence resonance energy transfer, and dynamic quenching determine the excited-state lifetime of a fluorophore. The FLI technique provides a way to measure these processes directly at 103–106 pixels in an image. In addition, if image domains differ with respect to the mean fluorescence lifetime, FLI can be used to improve the contrast of a fluorescence image. By measuring the fluorescence lifetime one can determine whether fluorescence intensity differences from different locations in an image can be attributed to differences in dye concentration or whether physical spectroscopic effects such as local differences in the rate of dynamic quenching are responsible. All the above applications provide new possibilities for biology and medical diagnostics. However the speed of data acquisition and analysis in current FLI instrumentation is limited in general to several minutes; for real-time applications (in order to follow rapid changes of microscopic samples or make in vivo endoscopic medical diagnosis) the present instruments are too slow. We present here a FLI apparatus that is capable of acquiring, processing, and displaying fluorescence lifetime-resolved images in quasi-real time. We also present rapid algorithms for analyzing the data in real time.

A high speed multifocal multiphoton fluorescence lifetime imaging microscope for live-cell FRET imaging

“…Whilst wide-field fluorescence lifetime imaging is possible at up to video frame rates with gated image intensifiers [54,55,56], this is not practical in a biological setting due to sample limitations (i.e. excited state fluorophore saturation), significant imaging artefacts and excitation photon flux that may be damaging to cells [19,26,27,29].…”

Spectrally resolved fluorescent lifetime imaging

“…The frequency-domain method most commonly used for the measurement of lifetime images involves exciting a sample with a sinusoidally modulated light source, such as a laser (Marriott et al 1991;Schneider & Clegg 1997;Esposito et al 2005) or LED (Dinish et al 2006;Elder et al 2006), and detecting with a modulated detector. The most common implementation is the homodyne approach in which both excitation source and detector are modulated at the same frequency and a series of images are collected as the phase between light source and detector are shifted.…”

Suppression of photobleaching‐induced artifacts in frequency‐domain FLIM by permutation of the recording order

“…For every pixel, the fluorescence intensity as a function of the phase difference between excitation light and image intensifier gain is determined by recording a sequence of images. The conventional way of determining phase and modulation depth from these recorded images is by Fourier analysis (9,10). Assuming measured phases to be spread equidistantly over the 360°range, F sin , F cos , and F DC , are determined for every pixel using: where K represents the number of recorded images, I K the intensity in the kth image, and n the harmonic of interest.…”