http://confocal-microscopy-list.275.s1.nabble.com/image-brightness-on-a-laser-scanning-confocal-tp7581050p7581051.html
ago. The gist of it was that magnification is pretty much completely
irrelevant unless the image is printed. NA is king.
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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>