image brightness on a laser scanning confocal

Posted by Julio Vazquez on
URL: http://confocal-microscopy-list.275.s1.nabble.com/image-brightness-on-a-laser-scanning-confocal-tp7581050.html

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Dear confocalists,

I have been thinking about the relative brightness of different objectives on a point laser scanning confocal microscope. The formulas one finds in books, etc, report brightness as proportional to NA^2 / M^2 for transmitted light, and NA^4 / M^2 for epifluorescence. However, I have never seen a specific discussion of single point scanners vs widefield. It is my impression that on a point scanner, the laser beam is focussed to a spot whose radius depends on the NA; on the collection side, light emitted by the spot is collected back onto a PMT, with a collection efficiency also related to the NA. At neither stage does the magnification seem to play a role. This leads me to think that the brightness of an objective on a point scanner is proportional to the fourth power of the NA, and independent of the magnification. I did some quick and dirty measurements on 200 nm beads which seemed to support this (although complicated by various factors such as the size of the back aperture, which made it difficult to get precise measurements of excitation power at the sample with high NA lenses, for example, and other things). However, I am puzzled I have never seen mention of this anywhere. Is it correct that magnification is irrelevant for image brightness on a point scanner, or am I way off the mark?


Julio Vazquez
Fred Hutchinson Cancer Research Center
1100 Fairview Ave N., DE-512
Seattle, WA 98109

http://www.fhcrc.org/en.html