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STED (stimulated emission depletion) microscopy is a technique which enables sub-diffraction limit of light imaging.

The depletion laser is at 775nm, and about 500 mW, concentrated for a short sequence of pulses on an area about the size of ~0.2 um^2

As far as I can tell very few organic molecules, nor water, absorb in this range of 775nm.

So why does it cause photodamage? Thermal damage sure, but why bleaching fluorophores so fast?

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    $\begingroup$ It is good practice to define acronyms when they are first used. Please define “STED”. $\endgroup$
    – Ed V
    Apr 14, 2021 at 19:28
  • $\begingroup$ Edited for your comment. $\endgroup$
    – dlight
    Apr 14, 2021 at 19:41
  • $\begingroup$ Thanks! I cannot answer your question, but now it has higher likelihood of getting a valid and helpful answer from someone. $\endgroup$
    – Ed V
    Apr 14, 2021 at 21:14
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    $\begingroup$ @dlight, There are reviews on this topic. There are dyes which excited by red wavelengths and that is what STED uses, right? One of the article suggests "Photobleaching is primarily caused by the depletion light acting upon the fluorophores in the excited states." $\endgroup$
    – AChem
    Apr 14, 2021 at 21:51
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    $\begingroup$ See this one too: onlinelibrary.wiley.com/doi/full/10.1111/jmi.12698 $\endgroup$
    – AChem
    Apr 14, 2021 at 21:51

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Ballpark calculation: Wikipedia mentions the sun irradiation received at earth's sea level is up to about $\pu{300 W/m^2}$. This is after the atmosphere filtered out already much. You describe an experiment that where $\pu{0.5 W}$ set foot on about $\pu{0.2 µm^2}$ (or, equivalent to to this, $\pu{0.2 × 10^{-6} m^2}$).

This translates to a power density of

$$\frac{\pu{0.5 W}} {\pu{0.2 × 10^{-6} m^2}} = \pu{2.5 × 10^6 W/m^2}$$

which is more than 8000 times larger than above assumption. This one of the reasons why samples are excited by pulsed laser radiation with repetition frequencies in the range of MHz. And still degradation by photobleaching may occur, which one already tries to reduce, e.g., by excitation which do not hit the centre, but a tail of an absorption band.

(The mechanisms are different, but the results of exciting a small area with too much energy are similar to microscopic techniques based on second harmonic and complementary third harmonic generation (an example).)

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