Re: Size of a bleach point

Posted by Kate Luby-Phelps on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Size-of-a-bleach-point-tp7590808p7590812.html

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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