Time resolved fluorescence basically gives us the time taken by an electron to de-excite to the ground state followed by the excitation pulse. And since this process is random in nature we get a histogram and then we get a decay profile with which we can determine the lifetime and other important information. In reverse start stop TAC, the photon pulse from the detector (photomultiplier tube) starts the time sequence and the sync from laser stops TAC. So basically here we are calculating the time between the detected photon pulse and the next laser pulse (whereas lifetime = time between excitation and deexcitation pulse). My question is how does this time correspond to the time taken by a photon to de-excite from the excited state?
In a TCSPC setup you are basically measuring the time difference between a start and a stop events. Hence, if you swap the inputs, you just reverse the time axis. For a proper acquisition you should monitor the TCSPC histogram accumulation in realtime, and adjust the delays between start and stop in order to have the full decay within the full-scale range (FSR) of the TAC. In other words you need to be sure your laser sync signal arrives *after* the detector pulse, and that the time-difference falls within the TAC range.
Once your setup is configured, you need to reverse the time axis on the acquired TCSPC histogram to get the decays in the conventional direction.
Saurabh,
May I suggest a text that you should find very useful. It is out of print, but you can get used copies of it through Amazon.
"Time-Correlated Single Photon Counting" by Desmond O Connor and David Philips.
Another text also out of print, but still obtainable:
"Chemical Applications of Ultrafast Spectroscopy" by Graham Fleming
The second text covers more general aspects of Ultrafast Spectroscopy (ca. 1986), but includes TCSPC.
Cheers,
John
Have a look at a book chapter my supervisor wrote in 1991, you should get it on SpringerLink:
http://link.springer.com/chapter/10.1007/0-306-47057-8_1
We also have a newer chapter here which is a bit shorter:
https://www.researchgate.net/publication/257599541_Nanometrology
For TCSPC conditions are set so that a hit rate is less than 2 %. This is used to prevent photon pile-up. i.e. to prevent the probability of 2 photons hitting the detector. If this happens (at higher hit rates) the detector will only detect the first photon hence biasing the decay to short times.
Reverse mode is just used when the deadtime >> TAC range. You essentially are measuring the same decay just in reverse but speed up acquisition because instead of waiting for deadtime 100 times, you only wait for it 2 times. i.e. 100×1 µs vs 2×1 µs.
Older systems such as the HORIBA FluoroCube/TemPro typically had ~1 µs of deadtime so reverse mode was used for time ranges of 50 ns - 200 ns. Newer systems such as the HORIBA DeltaFlex/DeltaPro have a deadtime of
The bh TCSPC handbook is a great resource as well:
https://www.becker-hickl.com/literature/documents/flim/the-bh-tcspc-handbook/
And it's available for free.