Posted by
Moulding, Dale on
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Hi Xavi,
I tend to use the PSF generator in Imagej / Fiji.
http://bigwww.epfl.ch/algorithms/psfgenerator/You can take the full width at half maximal to indicate the bleach area. In xy and z. I tend to think of bleaching a volume rather than an area.
I'm sure this isn't perfect, as the energy isn't equally spread within the PSF. If I'm teaching this, I use the plot profile on live view of a PSF, fix the axes ranges on the graph, and run through the z stack to show the energy distribution in different z slices.
Or save a small (65x65x65 pixel) PSF with 50 or 100 nm xyz pixels, and use ICY 3D viewer with a colour map (LUT) like Parula. I always need to fix the metadata in ICY to the correct XYZ dimensions.
cheers
Dale
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Sent: 23 April 2020 23:21
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Subject: Size of a bleach point
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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.
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*Xavier Sanjuan*
Advanced Light Microscopy Unit
Parc de Recerca Biomèdica de Barcelona
Doctor Aiguader, 88
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