Is there any difference between time-correlated single photon counting (TCSPC) and Fluorescence Lifetime Imaging microscopy (FLIM)? TCSPC i.e. lifetime measurement of the fluorophore is basically done in a solution whereas FLIM is performed by drying the same sample on a cover slide and then data is recorded.
What is new in FLIM technics compared to TCSPC in the objective to make images and videos. TCSPC is one of the four technics to measure luminescence lifetimes. The other three are phase fluorimetry, streak cameras and boxcar light collection. FLIM can be done by TCSPC (scanning or scanningless) or phase fluorimetry or with pulsed light amplifyers.
FLIM measurements are usually carried out using TCSPC or Frequency Domain methods. The Phasor method is becoming very popular to analyze FLIM data and it can be carried out using either time or frequency domain approaches.
Thank you for your warm response@Robert Bernard Pansu and @David M Jameson. However, my question is basically related to the lifetime components derived from TCSPC and FLIM data. In most cases, the data for the same system obtained do not match each other. Can you kindly reflect on this point?
In principle the lifetimes obtained by either method should match. I attach a figure referring to the paper by Boens et al which compares lifetimes from samples sent to different labs using either TCSPC or frequency domain methods and they were all in agreement.
If you are analyzing data sets on the same system obtained with each method and get different components then the issue lies in the data acquisition and analysis. In such cases the details of the data acquisition are important. Specifically, if you are trying to analyze a complex emission with only one modulation frequency you have a real problem. To compare the two methods fairly you need to use a range of frequencies - unfortunately most FLIM instruments only use one frequency. This problem is why most people now use phasor analysis which can be done on both time domain or frequency domain data. I also attach a file which reviews this method.
Thank you David M Jameson for your valuable response. I understand sir that lifetime data should match whether it's by FLIM or TCSPC and I agree with the fact that one must use a range of frequencies when comparing two methods. If both the techniques are performed in the solution phase i.e. for TCSPC the solution is placed in a cuvette and data is recorded and in the case of FLIM, the same sample solution can be drop cast on a dry cover slip, and then FLIM is performed, it can be expected that the lifetime data obtained are comparable. However, FLIM can also be performed by drying the same sample on a cover slide, and then the data is recorded. Also, since in FLIM we are exciting a particular point or our area of interest (if carried out in solid-state i.e. by drying) will it not vary with data obtained from the TCPSC method performed in the solution phase?
Hi Souvik - I do not actually understand your point. FLIM is most often carried out using TCSPC. I think that you may be confusing the FLIM with frequency domain (FD) measurements. In general though I would agree that carrying out either FD or TCSPC measurements the results could differ if one looks at a sample in solution compared to a solid state on a slide. Also the point under observation on a slide will affect the results. Sorry not to be clear but I do not really understand your point.
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