Fluorescence Lifetime and Fluorescence Resonance Energy Transfer. Image What is Fluorescence Lifetime? Fluorescence can be thought of as an energy transitions from the electronic ground state (S0) to its excited state (S1) in a molecule. This transitions is produced by incident light with the appropriate energy (eg light with a wavelength of 488nm has enough energy to excite GFP to S1). The fluorescent molecule stores the absorbed energy for a short time until it is emitted as fluorescence. The time a molecule spends in its excited state is known as the fluorescence lifetime. This time is usually nano-seconds (10-9s) for most of the fluorescent probes used in biological applications. Biological Uses for lifetime measurements Determination of the cellular environment containing the sample molecules such as pH, temperature, polarity. Measuring the distances between different parts of the same fluorescently tagged protein or different fluorescently tagged proteins (FRET measurements). What is Fluorescence Resonance Energy Transfer (FRET)? FRET as a techniques has been around for over 20 years with thousands of scientific papers published either developing it or using it in biological applications. Consequently the mechanics of FRET are very well understood. Image FRET describes the non-radiative transfer of energy stored in an excited fluorescent molecule (donor) to a non-excited different fluorescent molecule (acceptor) in its vicinity. Three conditions must be fulfilled for FRET to take place: Overlap of donor emission spectrum with acceptor excitation spectrum Molecules must be in close proximity (~10nm). Molecules must have the appropriate relative orientation FLIM-FRET Experimental Design Culture cells with proteins of interest expressing required fluorescent proteins (eg GFP and mCherry, which is what this protocol used) Check that the cells are expressing the fluorescent proteins in the correct localisation and that the cells are viable Culture cells for a positive and negative FRET control to test the system. Positive controls would be cells expressing a direct GFP-mCherry fusion, Negative controls would be cells expressing free GFP and free mCherry. Cells to use in study Cells expressing GFP fused to mCherry Cells expressing free GFP and free mCherry Cells expressing fused GFP Cells expressing protein of interest fused to GFP and free mCherry Cells expressing protein of interest 1 fused to GFP and protein of interest 2 fused to mCherry (the actual experiment sample) Image cells in Phenol free tissue culture medium Check that cells have correctly expressed the GFP and mCherry either on a standard widefield microscope or using the normal confocal settings (ie not the multiphoton). Note that a lot of multiphoton lasers don't go high enough into the red (>1100nm) to be able to excite the mCherry so this will have to be checked in standard mode. This doesn't matter for the experiment as all the lifetime measurements will be made using the GFP. Set up the multiphoton laser to excite at ~890nm, if using the external FLIM detector ensure that a bandpass filter of 500-550nm is installed. When using the internal FLIM detector set the spectrophotometer to 500-550nm. To optimise the image adjust the laser power, gain and offset on the MP laser for GFP until you get a good signal to noise and the cell is able to survive. This step may take quite a while and several different settings until the ideal is found. Its advisable to then save these settings for future use. This article was published on 2024-06-17
