Re: Tissue imaging auto fluorescence and bright dot-like signal

Posted by Lu Yan on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Tissue-imaging-auto-fluorescence-and-bright-dot-like-signal-tp7588326p7588346.html

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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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