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Cammer, Michael-2 on
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Plexiglas may not scatter & absorb light the same as your biological samples, but with SP2 AOBS and SP5 AOBS we bleached spots inside fluorescent Plexiglas and then went back and imaged Z series of the bleached volumes to see focal plane and cones above and below.
Two examples:
https://www.flickr.com/photos/mcammer/2605562876/https://www.flickr.com/photos/mcammer/2608080259/Michael Cammer, Sr Research Scientist, DART Microscopy Laboratory
NYU Langone Health, 540 First Avenue, SK2 Microscopy Suite, New York, NY 10016
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From: Confocal Microscopy List <
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Sent: Friday, April 24, 2020 9:29:14 AM
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Subject: Re: Size of a bleach point
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For quantitative analysis of recovery after bleaching a spot with a Gaussian laser beam (TEM00), the beam waist typically is used for the radius term. This is the width at which the normalized intensity of the beam drops to 1/e^2.
To measure the intensity profile of the beam, you would take an image of the parked beam on a uniform, photostable sample, use ImageJ to get an intensity profile across it and fit a Gaussian to the profile. In principle, you could also do this from the inverted intensity profile of the bleached region in the first image acquired after bleaching, but if recovery is fast, this will be an underestimate of the "true" radius of the spot.
The FWHM is approximated by 2.355 x the standard deviation of the Gaussian and the waist is about 1.7 x FWHM. The difference is important for quantitative analysis to obtain the transport characteristics of what you are bleaching because the area of the bleached region is significantly larger using beam waist than using FWHM.
Have fun!
Kate