Difference between revisions of "Photobleaching Model"

Revision as of 22:29, 7 July 2011

Trying to figure out a better model for the photobleaching of Sybr Green I in the DNA melting lab, because our current model fails at long times.

What is the current model?

The Matlab DNA melting simulation writeup has a fixed percentage of the active fluorophores being destroyed every time step. The bleaching coefficient B is set such that half the fluorophores will have been destroyed by the end of the simulation, if you had a constant input.

I think this makes at least some sense, because if you have the fluorescence be low and then go up at the end, then the bleached is more than half of the unbleached, because there were inactive fluorophores not being destroyed... argh I am confused.

How is it done in EstimateDnaMeltingParameters?

bleachingCorrection = (1 - bleachingCoefficient .* cumsum(thermalQuenchingCorrection .* dnaFractionVersusTime)).^bleachingPower .* ...
                      (1 - bleachingConstant .* (0:(length(dnaFractionVersusTime) -1))');

What are bleachingCoefficient and bleachingConstant?

How does Sybr Green actually photobleach?

Photobleaching is "a very poorly understood phenomenon" [1]. Remember, the other word for it is photodegradation.

To a first approximation, every fluorescence event has an equal chance of causing the destruction of that one fluorophor (Wikipedia). Also fluorophor lifetimes given in terms of avg number of photons they can emit before dying.

• Call Sigma Aldrich? Call Invitrogen/Life Technologies? Who all sells Sybr Green I? (Life Tech owns it and licenses to other companies. Molecular Probes is a sub-entity of Invitrogen.)

• Sigma's Sybr Green I datasheet.
  • Fluorescence quantum yield ~0.8
  • 497nm/520nm peaks

• Zipper et al [2] is the classic sneaky structure-finding paper. Also talks about the importance of the stoichiometric ratio (dbprs). Need to reread this carefully because last time I was paying attention to a different aspect.

• Henary & Mojzych [3] book chapter. Says Chen et al (ref 7) found first order photobleaching kinetics.

• Chen et al [4] says first order kinetics for a cyanine-family dye. Full text not available grrrr. So I don't know how long they treated the dye, or whether they really mean pseudo first order.

• Epling and Lin [5] find first-order kinetics in bleaching some dye using TiO2 as a catalyst, but no bleaching without catalyst. Unclear if this is applicable to us. Also, they seem to have used constant light input.

• Wikipedia on cyanine dye applications notes that the dye to basepairs ratio matters; above 1 dye molecule per 60 bases, you can start to see quenching. Oh, fuck. Do we have to account for this too? What dbprs have we been using? Does it matter if the DNA is ss or ds? (i.e. if you melt the DNA does the dbprs in the tube increase?) The Zipper paper talked about this...

• Ward & Marples [6]

"DNA-bound photobleached SYBR Green I is reported to lose its DNA-binding affinity and dissociate; free dye can then re-bind to DNA thereby restoring the fluorescence (Molecular Probes, Inc. personal communication). Consequently, it is possible to rescore images stained with SYBR Green I following photobleaching by storage in the dark and subsequent re-hydration." WHAT WHAT WHAT is this also definitely calling Molecular Probes tomorrow :) and maybe Ward & Marples too :) (Also, looking at their figure 1A, looks like they see first order kinetics too.)

• Widengren & Schwille [7] check out equation 5, it looks pretty much like what we had in the matlab page. But... their timescales are different, and their derivation rests on assumptions about timescales. In particular, because they're looking at a small volume, they assume that photobleaching and diffusion have the same time scale, which is not true for us. (Well, is it? What the hell is the timescale for photobleaching anyway?) Also, they're using an autocorrelation equation, which I don't really understand how it works.

Experiments

How to investigate photobleaching? Just shine light at samples for an hour? Expts done with fluorescein in past

I wonder if there's anything we can add to the solution to reduce photobleaching. Of course, it'll also affect melting, so I don't know if it's a good idea.