Spectral resolution in conjunction with polar plots improves the accuracy and reliability of FLIM measurements and estimates of FRET efficiency

Abstract

SummaryA spectrograph with continuous wavelength resolution has been integrated into a frequency-domain fluorescence lifetimeresolved imaging microscope (FLIM). The spectral information assists in the separation of multiple lifetime components, and helps resolve signal cross-talking that can interfere with an accurate analysis of multiple lifetime processes. This extends the number of different dyes that can be measured simultaneously in a FLIM measurement. Spectrally resolved FLIM (spectral-FLIM) also provides a means to measure more accurately the lifetime of a dim fluorescence component (as low as 2% of the total intensity) in the presence of another fluorescence component with a much higher intensity. A more reliable separation of the donor and acceptor fluorescence signals are possible for Förster resonance energy transfer (FRET) measurements; this allows more accurate determinations of both donor and acceptor lifetimes. By combining the polar plot analysis with spectral-FLIM data, the spectral dispersion of the acceptor signal can be used to derive the donor lifetime -and thereby the FRET efficiency -without iterative fitting. The lifetime relation between the donor and acceptor, in conjunction with spectral dispersion, is also used to separate the FRET pair signals from the donor alone signal. This method can be applied further to quantify the signals from separate FRET pairs, and provide information on the dynamics of the FRET pair between different states.

N-Way FRET Microscopy of Multiple Protein-Protein Interactions in Live Cells

“…This additional information should facilitate interpretation of changes in fluorescence signal amplitudes in terms of FRET efficiencies to improve distance estimates. Additionally, the use of these matrices may improve fluorescence lifetime imaging of FRET signals by facilitating the unmixing of overlapping fluorescence components [45].…”

Multidie LED combined with homogenizing optics to improve frequency response and intensity for FLIM applications

“…We used homodyning method to measure fluorescence lifetime in the frequency‐domain (Chen & Clegg, , ). Modulation of the same frequency () was applied to both the LED and the signal acquisition units.…”

Förster resonance energy transfer microscopy and spectroscopy for localizing protein–protein interactions in living cells

“…Camera-based FD FLIM is typically applied in widefield or spinning-disk confocal microscopes, using a LED or diode laser excitation light source and an image-intensifier camera, both modulated at slightly different frequencies (Heterodyne) (131) or at the same frequency (Homodyne) (132,133). Similar to the gating-camera TD FLIM, the camera-based FD FLIM technique also provides fast imaging speeds (54).…”