Hi all,
I have a theory question that is probably obvious to some of you!
I’m confocal scanning a FITC solution as follows: Imgur: The magic of the Internet with excitation at 520 nm and emission around 500 nm. I’m using time gating to eliminate laser reflection.
I thought I knew that fluorescence goes from lower wavelength excitation to higher wavelength emission according to Stokes, because a fluorophore producing fluorescence absorbs the higher energy photon, does some work thus losing energy in the process, and then emites a photon that must have a lower energy (higher wavelength) than the incoming one.
The anti-Stokes shift description on the Wiki page mentions a special case where the fluorophore receiving energy from a surrounding crystal lattice, but I’m scanning freely diffusing FITC molecules in water…
Please help me understand how I am receiving higher energy photons from FITC than the photons I’m pumping into it!
Thanks in advance.