Good single-molecule fluorophores must meet several criteria. To name a few, the molecules must have high quantum yields, must be photostable, with a large absorption cross-section and a low yields into dark triplet states. (Ideally, a single-molecule fluorophore would have some inherent reporter function, but that’s a story for another day.) There are several popular long-lasting, bright fluorophores out there (e.g. rhodamines, terylenes, Cy3); my research involves developing and characterizing new compounds for single-molecule cellular imaging, which means quantifying the criteria I listed above.
One measure of photostability is the total number of photons each molecule emits before it irreversibly photobleaches (Ntot,emitted). Because photobleaching is a Poisson process that depends on the number of times the molecule reaches the excited state, Ntot,emitted for a fluorophore should not depend on the laser intensity (unless you illuminate beyond the saturation intensity): at a higher intensity, the molecules will absorb and emit photons at a higher rate, but photobleach in less time, yielding the same number of photons than at a lower intensity. So the Ntot,emitted of a particular fluorophore in a particular environment tells you how bright or long-lasting the fluorophore is; the higher the number, the better the fluorophore (all else the same).
I’ve measured the Ntot,emitted for a fluorophore on the bulk and single-molecule level—experiments which require different analysis. For the single-molecule measurement, I record movies of a wide-field illumination of several molecules well spaced in a polymer film. Then I integrate the counts from each molecule and plot the distribution from over 100 individual molecules. The distributions look like this:
The distributions are exponential characteristic of a Poisson process. From this distribution, I can determine the average Ntot,detected and convert this to Ntot,emitted using the detection efficiency of my setup (typically ~10% for epifluorescence).
The bulk measurement is a little less conceptually obvious. I use the same setup, but overdope the dye into the polymer film, so the intensity of the entire field of view versus time in the movie looks like this:
From the exponential fit, you can extract the average time before photobleaching for the molecules. This value, combined with the absorption cross section and the laser intensity, can give you the number of photons absorbed per molecule. Using the quantum yield, you can then calculate the Ntot,emitted.
So what’s my whole point here? Given that the two experiments I describe use different measurements and calculate Ntot,emitted using different parameters, I was really happy to see that the calculated values of Ntot,emitted were very close from bulk and single-molecule experiments when I compared them directly. That’s all.