measuring total photons emitted

August 15, 2006 at 10:09 am | | everyday science, single molecules, tutorial

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:

sm_total_photons.jpg

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:

3-3_1__bulk_double_exponential.jpg

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.

[Update: You can find some of the relevant equations in this paper or the SI of this paper.]

7 Comments »

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  1. I think you owe Dr. Zare some royalties for that histogam.

    Comment by kendall — August 15, 2006 #

  2. Do you mean the Zare-o-gram?

    Comment by sam — August 15, 2006 #

  3. I will like to know what causes photobleaching of fluorophores when using a Confocal microscope. Is it the instantaneous intensity of the laser beam, the integrated intensity or the total laser energy that the sample is subjected to.

    Comment by jimmy — September 21, 2006 #

  4. jimmy, photobleaching is usually explained by a photoreaction of the fluorophore with triplet oxygen. So the chance of photobleaching depends on how often the molecule is in the excited state. The rate of absorption depends on the absorption cross-section of the fluorophore and the laser intensity (e.g. W/cm2).
    .
    Does that help?

    Comment by sam — September 22, 2006 #

  5. Is there a fluorescing molecule known which doesnot photobleach ?

    Comment by Priya — February 6, 2009 #

  6. not really. there are a few organic fluorophores that last hours when embedded in a crystal or at cryogenic temperatures. diamond NV centers last basically forever.

    Comment by sam — February 6, 2009 #

  7. […] 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. | | […]

    Pingback by Everyday Scientist » on-time is not enough! — March 20, 2009 #

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