Dear Samy,

I am also no expert in this technology but I know that Viacheslav O. Nikolaev is one. You should check his published literature, e.g.: FRET measurements of intracellular cAMP concentrations and cAMP analog permeability in intact cells; Nature Protocols volume6, pages427–438 (2011); doi:10.1038/nprot.2010.198

Best

Maks

It is possible to do live cell imaging with FRET. Are you doing acceptor photobleaching to measure FRET? If so, you will need to take a baseline of time lapse imaging of the donor alone (without acceptor bleaching) with your imaging conditions to see if there is bleaching before you go ahead with the bleach. You lose a lot of signal with a spinning disk and need to compensate with more excitation power which will accelerate bleaching.

Noise is also a big factor and will obscure FRET measurements no matter which type of FRET you do. You will probably be better off with widefield which will have several benefits. You can use lower light intensities and get more signal, and set the camera to lower gain and slower readout which will reduce noise. I think getting FRET with a disk will be tough.

Hi Samy, photobleaching is more prevalent in some fluorophores. Fluorescein, PE etc.

Atto dyes and quantum dots are more stable.

Dear Samy,

I would like to add to the discussion that you can use FLIM (Fluorescence-lifetime imaging microscopy) to measure FRET. FLIM can be done with different measurement techniques, for FLIM on a spinning disk you can use a camera-based technique. When FRET occurs the Lifetime drops. Widefield FLIM (or in combination with a Spinningdisk) is suitable for Time-lapse life cell imaging. It is possible to get even below second temporal resolution. In addition to Maksymilian Prondzynski suggested article, I think the following Nature Methodes article covers what you are looking for: Raspe M et al. "siFLIM: single-image frequency-domain FLIM provides fast and photon-efficient lifetime data." (DOI: 10.1038/nmeth.3836 )

Greetings,

Johan Herz