Re: wide field bead fluorescence

Posted by Reto Fiolka on
URL: http://confocal-microscopy-list.275.s1.nabble.com/wide-field-bead-fluorescence-tp7582644p7582647.html

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Hi David

Maybe because the extend of the PSF (widefield or confocal) is about 4 times larger axially
than laterally.

Thus the convolution of a shell (bead) with a cigar (PSF) might yield an intensity distribution
that looks a bit like a toroid.

Further, if your focal plane coincides with the center of the sphere, most signal will be
detected from the segments of the shell that intersect the focal plane or are close by. Since
the optical axis is tangential to the local shell surface there, more signal is integrated by the
axially extended PSF. The top and bottom part of the shell are already slightly out of the
depth of focus and will be detected more weakly. Even if they fall into the depth of focus,
the optical axis is normal to the surface there, and only a thin layer falls into the PSF.

Hence the drop off in the center.


Best,
Reto