http://confocal-microscopy-list.275.s1.nabble.com/Tissue-imaging-auto-fluorescence-and-bright-dot-like-signal-tp7588326p7588355.html
I confirmed with our sample prep guy, and our coverslips have been cleaned using similar protocol. But I will try to remind him about the bulk fluorephore solutions.
There are likely as many coverslip cleaning protocols as their are microscopists in the world.
Plasma cleaning for 10 minutes to an hour will do a lot. That alone is often sufficient.
The real cleaning protocol involves chemical treatment capable of etching glass. I prefer overnight in 2M KOH, but others advocate piranha solution (nasty, dangerous), chromic acid (worse), or HF (very very bad). Then sonicate in DI water for 30 min, maybe repeat that step, then sonicate in acetone and 100% EtOH. From there dry with dry + clean nitrogen or flame dry and then do the plasma cleaning step. Cleaned coverslips go into a vacuum dessicator or use immediately. Don't use glassware that has ever been near bulk fluorophore solutions - less of a concern for biologists but chemists in the area often have benches covered in fluorophore residue.
This cleaning protocol will get your coverslips clean enough to do quantitative single molecule imaging experiments with nearly no background.
> *****
> 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.
> *****
>
> Hi Alexander,
>
> Thanks for your reply. May I ask if you have any good way of cleaning
> those coverslips?
>
> Thanks,
> Lu
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:
[hidden email]]
> On Behalf Of Alexander Asanov
> Sent: Tuesday, June 05, 2018 3:30 PM
> To:
[hidden email]
> Subject: Re: Tissue imaging auto fluorescence and bright dot-like
> signal
> Importance: High
>
> *****
> 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.
> *****
>
> Hi Lu,
>
> We have seen similar with some coverslips. To exclude autofluorescence
> of the coverslip, you can use your 405 line or a 405 nm laser pointer
> to assess the autofluorescence.
>
> Best regards,
> Alexander N. Asanov, Ph.D.
> President, TIRF Labs
>
[hidden email]
> www.tirf-labs.com www.TIRFmicroscopy.com
>
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:
[hidden email]]
> On Behalf Of Yan, Lu
> Sent: Tuesday, June 5, 2018 3:10 AM
> To:
[hidden email]
> Subject: Re: Tissue imaging auto fluorescence and bright dot-like
> signal
>
> *****
> 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.
> *****
>
> Thanks you all for the information.
>
> We are trying different clearing and bleaching protocols, and so far
> had no luck in getting rid of it.
>
> I also noticed that lots of bead-like spots also showed up as I
> focused on the coverglass-sample interface, even without tissue sample or any sample.
> Those spots also have broad emission and show in almost all channels.
> It seems two separated issues, but I was wondering if it is possible
> that the dirt on the coverglass would get into the tissue and causes
> 'auto-fluorescence'?
>
> Thanks,
> Lu
>
> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:
[hidden email]]
> On Behalf Of Rusty Nicovich
> Sent: Friday, June 01, 2018 10:33 AM
> To:
[hidden email]
> Subject: Re: Tissue imaging auto fluorescence and bright dot-like
> signal
>
> *****
> 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.
> *****
>
> 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
> >
>
>
> ---
> This email has been checked for viruses by Avast antivirus software.
>
https://www.avast.com/antivirus>