Figure 3: Schematic of automated plate reader based on time-gated fluorescence lifetime imaging (FLIM). | Scientific Reports

Figure 3: Schematic of automated plate reader based on time-gated fluorescence lifetime imaging (FLIM).

From: Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

Figure 3

(A) The pulsed excitation light is selected with an appropriate filter from the “white light” emitted by an ultrafast supercontinuum laser source and enters the microscope either in a wide-field configuration or via a Nipkow disk unit to provide optical sectioning. The fluorescence is detected via a gated optical intensifier (GOI) that acts as a fast (~100 ps rise time) electronic shutter synchronised with the laser pulses. The GOI opens at various delays after excitation (e.g. t1, t2, t3) and intensity images are acquired with a CCD camera at each time delay, integrating for a few seconds. (B) Lifetime determination. The time-gated images (t1, t2, t3) are used to reconstruct the fluorescence decay of the fluorophore, which is analysed by fitting exponential decay functions, discriminating between the lifetime of the donor only (D only) and the lifetime of the donor undergoing FRET in the presence of the acceptor (D + A).

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