http://confocal-microscopy-list.275.s1.nabble.com/Tissue-imaging-auto-fluorescence-and-bright-dot-like-signal-tp7588326p7588353.html
Plasma cleaning for 10 minutes to an hour will do a lot. That alone is
glass. I prefer overnight in 2M KOH, but others advocate piranha solution
(nasty, dangerous), chromic acid (worse), or HF (very very bad). Then
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
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>
> 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
>
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>
> 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
>
> *****
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>
> 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
>
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>
> 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:
>
> > *****
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> >
> > 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
> >
>
>
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