Sergio provides support on all aspects related to optical microscopy, from image acquisition to image processing.
He is interested in helping researchers to master microscopy imaging techniques and to assist them in collecting and interpreting high-quality data. He has significant experience in Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM).
FRET is a process by which excitation energy is transferred from a donor molecule to an acceptor molecule. This process does not involve the emission of a photon and is highly sensitive to the distance that separates the donor and acceptor molecules. The latter property of the FRET process has been extensively exploited to study phenomena that result in sub-microscopic (i.e., less than 10 nm) changes in distance, such as protein conformational changes, the coming together of interacting biomolecules, changes in DNA structure, etc. In FRET microscopy, each location in the image has a FRET efficiency associated with it.
In a FLIM image, the contrast is provided by differences in the fluorescence lifetime of the probe. In other words, by differences in the average amount of time that the fluorescent probe spends in the excited state. The main advantages of FLIM are;
- The fluorescence lifetime is largely independent of the concentration of the fluorescent probe
- The fluorescence lifetime can be sensitive to the presence of other molecules of interest (e.g., a FRET acceptor) as well as to local variations in pH, polarity, viscosity, etc
In addition, when combined with FRET, FLIM can simplify the process whereby the FRET efficiencies are calculated, among other things by reducing the number of necessary controls.