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I've never tried to do this, but could one not use standard volumes of a standard dye concentration, for example in a multiwell slide?

                                                                                     Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of Sam Lord
Sent: Friday, 13 March 2015 5:41 AM
To: [log in to unmask]
Subject: Re: Photobleaching Standard

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Hi Neil,

Great question.

On Wed, 11 Mar 2015 14:38:28 -0400, Neil Anthony <[log in to unmask]>
wrote:
>I would presumably
>calculate the intensity decay over a time lapse as a metric, but wasn't 
>sure if the sample type was a factor as I don't know much about the 
>photochemistry.

I have a pet peeve: when a paper reports photobleaching half-lives or bleaching decay times *without accounting for the rate of absorbing photons*. 
Photobleaching is a statistical process that typically occurs from the excited state of the molecule (e.g. from intersystem crossing to a triplet state and then further excitation and photodegratation) or from an excited producing singlet oxygen that then reacts with that molecule or a nearby molecule.

Either way, the rate of photobleaching is going to depend on how frequently the dye cycles into the excited state. If you compare two samples without taking into account the excitation rate, you might think one sample is more susceptible to bleaching when really it is just brighter.

I recommend using the photobleaching quantum yield as an absolute measure. It is the probability of photobleaching with each photon absorbed, or the ratio of the bleaching rate to the photon absorption rate. It is easy to calculate if you know the extinction coefficient of the dye and the irradiance and wavelength of light you're using. See equation 2.1 of my paper here:

Thompson, M. a, Biteen, J. S., Lord, S. J., Conley, N. R., Moerner, W. E. (2010). 
Molecules and methods for super-resolution imaging. Methods in Enzymology, 475(10), 27-59. doi:10.1016/S0076-6879(10)75002-3 http://everydayscientist.com/pdfs/sjl16.pdf

Alternatively, maybe a more practical metric would be to change the illumination so that each sample has the same brightness on the camera or the same signal to noise ratio or some other useful quantity. Then record the bleaching half-time. That would tell you exactly what you really want to know: given a certain starting image quality, how long can I observe my sample? This measure should indeed scale with the photobleaching quantum yield, but it also takes into account the "brightness" 
of the dye (i.e. the fluorescence quantum yield times the extinction coefficient), as well as microscope factors like the chromatic aberrations and camera quantum efficiency at different wavelengths.

This is a pragmatic measure, but it would be a little hard to directly compare completely different dyes, samples, microscopes, etc. That's why I like absolute metrics, like the photobleaching quantum yield and dye brightness.

For a great reference on dye brightness, see:

A guide to choosing fluorescent proteins http://www.tsienlab.ucsd.edu/Publications/Shaner%202005%20Nature%20Method
s%20-%20Choosing%20fluorescent%20proteins.pdf

(Note the measure that Shaner used for photo stability does indeed take into account absorption rate: "Time for bleaching from an initial emission rate of 1,000 photons/s down to 500 photons/s (t1/2; for comparison, fluorescein at pH 8.4 has
t1/2 of 5.2 s).")

The reason Tsien uses t1/2 (AKA photobleaching half-life) is to avoid the pitfalls of multi-exponential fitting and reporting weighted decay constants (see Lakowicz or equation 2.2 of my paper above). Half-life is a super simple and pragmatic measure of sample photobleaching.

>On that note, can anybody recommend a good reference that discusses the 
>subtleties of photobleaching?

As far as I know, there is no single review that really encompasses all the subtleties of photobleaching. A lot of those subtleties are not well understood. 
People often blame singlet oxygen, but it's way more complicated than that (e.g. 
triplet/ground-state oxygen is an excellent triplet quencher, so it is actually good to have around to reduce dyes in the triplet bottleneck; see Hubner. J. Chem. Phys. 
2001, 115, 9619). There are many paths to photobleaching, they are difficult to observe, they yield a huge range of photoproducts, and each dye is likely to have a completely different way it prefers to die.

There are some great examples of papers where the authors discovered the photobleaching path of a particular dye or subset of dyes:

Dempsey GT, Bates M, Kowtoniuk WE, Liu DR, Tsien RY, Zhuang X. (2009). 
Photoswitching mechanism of cyanine dyes. Journal of the American Chemical Society, 131, 18192-3.

Dickson RM, Cubitt AB, Tsien RY, Moerner WE. (1997). On/off blinking and switching behaviour of single molecules of green fluorescent protein. Nature, 388, 355-8.

Toutchkine A, Nguyen DV, Hahn KM. (2007). Merocyanine dyes with improved photostability. Organic Letters, 9, 2775-7.

Kong X, Nir E, Hamadani K, Weiss S. (2007). Photobleaching pathways in single- molecule FRET experiments. Journal of the American Chemical Society, 129, 4643- 54.

Cordes T, Vogelsang J, Tinnefeld P. (2009). On the mechanism of Trolox as antiblinking and antibleaching reagent. Journal of the American Chemical Society, 131, 5018-9.

Berglund AJ. (2004). Nonexponential statistics of fluorescence photobleaching. The Journal of Chemical Physics, 121, 2899-903.

Best,

-Sam