Need assistance in calibrating the system using a Urea crystal - we are unable to obtain an IRF response. Anyone have suggestions on how to prepare the sample and get a response?
We have good agreement in our decay times for Coumarin 6 fluorescence (~2.5 ns), and can measure FRET activity in our positive control cells, but need/are supposed to measure the instrument response function to accurately fit our FRET samples with a biexponential decay. We are using an older version of the Picoquant software. Thanks!
You can get the IRF via any material that shows second harmonic generation, since the pulse from second harmonic generation will be of the save duration as the pulse going in (about 150fs), which is way shorter than the FLIM router can measure (normally 150ps). Therefore, you will get a pure IRF.
For second harmonic generation (SHG), we mounted 4-methyl-2 nitroaniline in permount to make permanent SHG slides. Just be careful and keep the laser power VERY low, as 4-methyl-2 nitroaniline is highly efficient at SHG, and will generate a very bright signal.
In our experience, we have never had to model the IRF, as we simply look at the decay histogram from 1ns out to 8ns, therefore only analyzing the data outside the IRF.
If you are doing FLIM-FRET, we have found that a very robust way to prove FRET, is to first measure the lifetime of the donor via FLIM. Then perform acceptor photobleaching, and look for the increase in fluorescence. Then go back and re-measure the lifetime via FLIM. If the sample is truly undergoing FRET, then it will have a reduced lifetime on the first FLIM run, then will increase in brightness during acceptor photobleaching, and then will show a fully recovered lifetime on the second FLIM run.
We have found that you can get false positives in FLIM, by having a convolution of multiple lifetimes (this can be sorted out using phasor-FLIM, but is a bit more complicated). You can also get false positives in acceptor photobleaching, where the photobleaching can sometimes actually lead to an increase in autofluroescence. However, between FLIM and acceptor photobleaching, these false positives are mutually exclusive, and therefore you can be certain you have FRET.
Thank you all very much for your responses. Your suggestions are very helpful and I will look into these alternative solutions (and photobleaching!) for my IRF and FRET measurements.
Incidentally, we are using a two-photon laser, and I have consulted the Picoquant support page many times, but thank you for the suggestion Peter.
Janet
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