Posted by
George McNamara on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Size-of-a-bleach-point-tp7590808p7590811.html
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Hi Xavi,
For fastest photobleaching, generally best to use maximum power for as
brief a time as possible. The more photons, the bigger the spot. Oxygen
radicals and dye radicals diffuse short distances relative to PSF
(nanometers ... though activated tyramide radicals have a diffusion
radius of ~100 nm, and can be restrained by increasing viscosity or
additives ... cytoplasm is higher viscosity and density than water ...
so: radicals diffusion radius might be suppressed in live cells that are
[over]expressing catalase).
A(nother) good PSF calculator is available free at SVI
https://svi.nl/NyquistCalculatorMicroscopy Nyquist rate and PSF calculator.
I liked this recent publication on the photophysics and photochemistry
of fluorescence - and generalization across the UV-Visible fluorophores:
Aleksandr Barulin and Jeroome Wenger 2020
Ultraviolet Photostability Improvement for Autofluorescence Correlation
Spectroscopy on Label-Free Proteins
J. Phys. Chem. Lett. 2020, 11, 2027−2035
https://dx.doi.org/10.1021/acs.jpclett.0c00209 From abstract: "Our results show that the underlying photochemical
concepts initially derived for organic visible fluorescent dyes are
quite general."
George
p.s. tyramide references:
* Chen ... Belmont 2018 J Cell Biol
https://doi.org/10.1083/jcb.201807108* Phil Moens, pers. comm. [1993?] (ex-NEN, before acquisition by
PerkinElmer and Phenoptics division spinout to Akoya).
On 4/23/2020 6:21 PM, SANJUAN SAMARRA, XAVIER wrote:
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>
> Dear listers,
>
> When using the Bleach Point feature in the Leica confocal systems (I'm sure
> it also exists in the other brands), I don't know how to measure the
> surface of the bleached area. I figure out the wavelength of the laser beam
> and the NA of the lens affect the size of that area, so would it be correct
> to use the resolution formula to calculate it? Or am I mixing up things
> here?
>
> Thank you very much,
>
> Xavi.
>
> ___________________________________
>
> *Xavier Sanjuan*
> Advanced Light Microscopy Unit
>
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