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The Bell Single Molecule and Fluorescence Lab
Advanced Characterization of Fluorophores
A temporal histogram of the fluorescent decay of a fluorescent solution recorded using the time-correlated single photon counting (TCSPC) instrumentation. Interestingly, in most solvents, a decay with lifetime of approximately 4 ns is seen, like in the ethyl acetate (EtOAc). However, in dimethylsulfoxide (DMSO), this decay is highly quenched, giving a biexponential decay with lifetime.
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A temporal histogram of the fluorescent decay of a different molecule, however this time at the single molecule level. By combining the isolating power of our confocal microscope with the stopwatch abilities of our TCSPC, we’re able to get temporal information at the single molecule level. In this case, we notice that the single molecule lifetime of the molecule was 6.17 ns, which agrees with the lifetime of the bulk sample which was on average 6.13 ns.
The fluorescence emission profile of a single molecule. Using confocal microscopy, we can collect the emission profile of a single molecule at a time, and collect information that might be lost due to ensemble averaging. As can be seen when compared to the bulk emission profile, this particular molecule emitted light that looks a little more green to the eye than the bulk sample does. However, not many of the singular molecules emit light like this, and so in a bulk sample, most of the fluorescence will look yellow.