What techniques can be used for FRET quantification?

Techniques that can be used for FRET quantification include:

• Acceptor photobleaching: Acceptor photobleaching involves quantifying the rise in donor emission subsequent to photobleaching of the acceptor.
• FLIM: FLIM allows researchers to map both the spatial and temporal variations in the lifetime of fluorescent molecules within a single living cell, going beyond decay curves. 
• Sensitized emission: In sensitized emission, the donor fluorophore is excited, and the resulting signal is captured using emission filters designed to detect both donor and acceptor fluorescence. 
• Spectral imaging: Spectral imaging involves exciting the sample using one or two specific wavelengths and then capturing the entire spectral profiles of both the donor and acceptor fluorescence. 
• Fluorescence polarization imaging: Fluorescence polarization imaging involves measuring fluorescence polarization both parallel and perpendicular to the excitation light, ensuring high signal-to-noise ratios.