Posted by
Rusty Nicovich on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Tissue-imaging-auto-fluorescence-and-bright-dot-like-signal-tp7588326p7588335.html
*****
To join, leave or search the confocal microscopy listserv, go to:
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopyPost images on
http://www.imgur.com and include the link in your posting.
*****
Sounds exactly like lipofuscin. We are deep into FISH imaging in mouse
brain and deal with this routinely. It's much worse in human and in
specimens from older donors.
Chemical treatment that Tim mentioned does do some good. It can reduce
desired fluorescence signal as well so check to see if your signal-to-noise
increases. Clearing your tissue with 8% SDS or similar can also attenuate
the lipofuscin signal. Then of course you can use the spectral signature
of the lipofuscin (and to some extent, its spatial signature vs
diffraction-limited RNA spots) to mask out regions where that signal
confounds the desired ones.
Thanks,
Rusty
On Thu, May 31, 2018 at 8:38 PM, Yan, Lu <
[hidden email]> wrote:
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
http://www.imgur.com and include the link in your posting.
> *****
>
> Dear listers,
>
> I have been imaging mouse brain tissue samples for the last a few days
> using multiple wavelengths laser as illumination (from 405 to 750 nm).
> Initially, the targeted genes in the brain slice were labeled with
> different dyes, e.g. Cy3, Cy5, and Cy7 etc., and I observed quite bright
> dots in corresponding channels, and thought they were RNA molecules.
> However, further examination showed that those dots showed up even in
> 'plain tissue' samples (no probes at all), and they virtually showed up in
> almost all fluorescence channels.
>
> The intensity was quite high that we could not distinguish the fluorescent
> probes signals. The plain tissue was snap-frozen and sectioned, and fixed
> with just methanol on coverglasses. I had tried different part of brain,
> different mouse, and they all showed similar results, i.e. bright dots
> cross the tissue sample, sometimes dense, sometimes sparse. They also
> distributed along the z axis, over about 10 um which is the sample
> thickness. In some region, I can see larger clusters giving much stronger
> signal. I also tried mouse liver samples, and the signal level was very
> similar to those from brain samples.
>
> I was wondering if anyone had similar experience in imaging tissue
> samples. What could be causing this kind of signal? Any input is welcome
> and appreciated.
>
> Thanks in advance,
> Lu
>