Does 2x scan speed + 2x averaging reduce photobleaching compared to 1x scan speed + 1x averaging (no averaging)?

Hi all,

New to this forum and excited to have discovered it!

I’m wondering if the statement in this post title is correct:

If I set up a typical CLSM line scan with 2x averaging, but 2x the scan speed, does that reduce the amount of photobleaching over the same amount of acquisitions, as compared to 1x averaging at 1x the scan speed?

By “amount of photobleaching” I suppose I mean the proportion of molecules destroyed after the acquisition has been finished.

Intuitively, yes - you don’t “pester” the molecules with as many photons per second, and give them a chance to recover. However, thinking once again, whether the molecule enters a triplet state does not depend on the photon dose i.e. scan speed, but is a chance process upon excitation. In the triplet state, whether it suffers chemical attack finally leading to destruction, is also a process independent of being bombarded by more or fewer photons.

I have not demonstrated this and feel like the statement is a “factoid” - not true, which I may have inadvertently been repeating.

Many thanks for any thoughts!

Hey @JelleP,

This general question of how illumination regime relates to photobleaching is still one with rather little hard empirical data behind it, but opinions on the topic are absolutely repeated all the time. To make matters worse, the few papers that have delved into very specific aspects of the photophysics here have often been referenced poorly and/or out of context. suffice it to say, it’s a super complicated topic, and I think most answers you’ll hear on this topic are largely folksy opinions stemming from uncontrolled observations.

I gave a longer response to this question in a previous issue:

I think the (theoretical) answer to your specific question hinges very much on the illumination power. At the extreme end of things, with very low excitation flux, i think most photophysical papers would suggest that you’re in a steady state anyway, and that the little “recovery” that the fluorophores would receive between scan 1 and scan 2 is negligible because the photon flux during the dwell is already so low. At the extreme high end of illumination flux, as you approach ground state depletion or more, things do indeed get interesting (as shown in Donnert et al 2007, in the linked comment) but, it’s mostly only been demonstrated for femtosecond pulsed lasers (not for CW lasers like on a point scanner) at very high illumination doses (many MW/cm2).

Hi @talley , thanks for the pointers to get started on this seemingly complex matter!

It looks like, for me, there is a lot more to read about photobleaching - I will have to have a good look at the materials you linked and therein. A cursory google search has been unsatisfactory and your post about resonant scanning already provides more background material than I could find myself. Thank you.

For example, I had totally not considered ground state depletion… There’s a lot going on.

I can imagine results from pulsed laser setups are more readily available, because under cw all these populations of molecules in different states mix together in time.

We have a first generation 80 MHz WLL :thinking:

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One more general comment:

I too think it’s super interesting to learn about photophysics and to hear some theories about why various illumination regimes might affect photobleaching. But at the end of the day, it’s also ok just to know if or that it has an effect on your specific instrument/fluorophore.

It’s very hard to do properly controlled studies to get at the why: but it’s less hard to test a particular configuration on your specific setup to answer “whether” on set of parameters reduces photobleaching. And practically speaking, thats what ultimately matters for your experiments :slight_smile:

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