on-time is not enough!

March 20, 2009 at 9:05 am | | single molecules, tutorial

jablonksi-diagramI’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.


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  1. Hi,

    Nice blog. It is nice to see that sufficiently engaged as to create a science blog!

    More on point, the things you discuss are part of the literature on blinking in quantum dots. Frankly, the points you correctly mention should be better discussed in any single particle analysis, whether molecules or quantum dots.

    In dots, one sees “flickering” – the dimming you mention. This phenomenology is clearly related to rates of absorption/emission. You can easily calculate these rates and you will find that there are many relevant processes happining during your binning time. Hence you are not really seeing a clean process. This situation strikes me as a pretty serious problem, at least for single dot experiments.

    In my group, we do femto experiments in which case the elementary processes are more cleanly discerned. But I have found that many of the elementary steps can yield insight into the phenomenology of single dot experiment where it appears that things are happening faster than binning times.

    Pat Kambhampati

    Comment by Pat Kambhampati — March 20, 2009 #

  2. Thanks for the comments!

    Comment by sam — March 23, 2009 #

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