Re: Are lower magnification objectives brighter?

Posted by Stanislav Vitha-2 on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Are-lower-magnification-objectives-brighter-tp7592013p7592041.html

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Assuming the beam is properly expanded, in a point scanning confocal system the area illuminated (the Airy disc) is only determined by wavelength and the NA of the objective.

Stan
Texas A&M University
Microscopy and Imaging Center

On Fri, 26 Mar 2021 08:17:05 +0000, Andreas Bruckbauer <[hidden email]> wrote:

>Hi Arnaud,
>
>make sure you have the same
>power at the sample plane with a power meter
>
>You also need to take into account the area which is illuminated.  When you change to lower magnification objectives with the same NA, you will illuminate a much larger area, so the power density goes down. This is an important factor for the fluorescence intensity, the number of excitation photons per fluorophore determines the number of emitted photons, if you spread the excitation fluorophores more thinly, you will get less fluorescence. If you just measure the LED power through the objective, you will miss this point. The question is, if you want to count this as "brightness" of the objective or not as you can just change the LED power or illumination beam path. 
>best wishes
>Andreas
>