I’ve seen a few papers recently that attempt to characterize the effect oxygen scavengers or triplet quenchers have on the photostability of single molecules. The main parameters they measure and compare across systems and fluorophores is the “on time”—the average time single molecules are fluorescent before they photobleach—and “off time”—the time spent in transient dark states.
Here’s my question: What about the emission rate? It’s not enough to report and compare only times. Photostability (either regarding bleaching or blinking) is related to a probability that a molecule will enter a permanent (or transient) dark state for every photon absorbed. The “on time” is only relevant when you also know the rate of photon absorption.
Moreover very fast excursions into transient dark states (i.e. triplet lifetimes are typically us or ms—much faster than the camera integration time) will appear as a dimming of the fluorescence, a decrease in the photon emission rate. By removing molecular oxygen (an effective triplet quencher) from solution, fluorophores often become dimmer because the triplet lifetimes increase. Thus, removing oxygen might make your dye last a longer time, but at the expense of brightness. This could more effectively be acheived by just turning down the excitation intensity (with lower background, too)!
So it makes me want to pull my hair out when I read long, detailed articles in prestigious journals that fail to mention even once the rates of photon absorption or emission when they report “on times” as photostability parameters.
Total number of photons is a more fundamental measure of photostability, but it still not enough for the full picture (it doesn’t report on blinkiness, for instance). Total photons plus “on times” is sufficient; or “on times” and emission rates; or enough variables to define the frikkin’ system.
UPDATE: Here’s a good paper that tests specific hypotheses about number and duration of off times. On the Mechanism of Trolox as Antiblinking and Antibleaching Reagent. JACS 2009, ASAP.