Re: Strange artifact in z series

Posted by Kurt Thorn on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Strange-artifact-in-z-series-tp592967p592971.html

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By bottom do you mean the part of the cell closest to the coverslip or
furthest from the coverslip?

It's not uncommon to see brighter fluorescence closer to the coverslip
than farther away from the coverslip as both scattering and spherical
aberration increase the further into a sample (i.e. from the coverslip)
one images.  Microscope objectives are corrected to give the best images
immediately adjacent to the coverslip and their optical performance
decreases the further into the sample you have to image.  Careful
matching of refractive indices of immersion media to the sample and
correction collars can minimize these effects.

Kurt

Pedro Camello wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi all,
>
> while performing z sections of mitochondria-stained living cells, I´m
> finding that the bottom part of the cell displays a brighter mitochondria
> fluoresecence, showing more contrast respect to cytosol than mitochondria in
> the top part of the cell. My system scans z from bottom to top. If it were
> simple bleaching due to laser light passing through the cell, I should
> observe that the bottom mitochondria are also less stained in a second
> series, but that´s not the case (the difference between top and bottom still
> there)
> Any idea to explain it?
>
> Thanks
>
> Pedro
>
>  

--
Kurt Thorn, PhD
Director, Nikon Imaging Center
University of California San Francisco

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