Tissue imaging auto fluorescence and bright dot-like signal

[Lu Yan]

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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

[Jacqueline Ross]

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Hi Lu,

My guess would be that it could be lipofuscin. I have observed this in human brain and mouse brain tissue previously. Also in heart tissue but haven't looked at liver. Lipofuscin fluoresces in the green/yellow-red emission range with a broad absorption spectrum as well as emission. In the human brain tissue, the spots look like fatty droplets and are usually adjacent to the nucleus, often in clumps.

Kind regards,

Jacqui

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Yan, Lu
Sent: Friday, 1 June 2018 3:38 p.m.
To: [hidden email]
Subject: Tissue imaging auto fluorescence and bright dot-like signal

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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

[timotheelionnet]

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That would be my first guess too. Sudan Black or True Black might help
mitigate this.
Tim
--
Timothee Lionnet - Assistant Professor of Cell Biology
Institute for Systems Genetics
NYU Langone Medical Center
Science Building, room 807
435 East 30th Street
New York, NY 10016
[phone]
http://www.timotheelionnet.net/




On 6/1/18, 12:02 AM, "Confocal Microscopy List on behalf of Jacqueline
Ross" <[hidden email] on behalf of
[hidden email]> wrote:

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>6LGCSskMZN4SHVGQ2eqR2SBLR9EUHN9Acbcc&e= and include the link in your
>posting.
>*****
>
>Hi Lu,
>
>My guess would be that it could be lipofuscin. I have observed this in
>human brain and mouse brain tissue previously. Also in heart tissue but
>haven't looked at liver. Lipofuscin fluoresces in the green/yellow-red
>emission range with a broad absorption spectrum as well as emission. In
>the human brain tissue, the spots look like fatty droplets and are
>usually adjacent to the nucleus, often in clumps.
>
>Kind regards,
>
>Jacqui
>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[hidden email]]
>On Behalf Of Yan, Lu
>Sent: Friday, 1 June 2018 3:38 p.m.
>To: [hidden email]
>Subject: Tissue imaging auto fluorescence and bright dot-like signal
>
>*****
>To join, leave or search the confocal microscopy listserv, go to:
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>Y&e=
>Post images on
>https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFAg
>&c=j5oPpO0eBH1iio48DtsedbOBGmuw5jHLjgvtN2r4ehE&r=pciiS70tK96NvFuBhwvhx_Iio
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>6LGCSskMZN4SHVGQ2eqR2SBLR9EUHN9Acbcc&e= 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

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[Rusty Nicovich]

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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:

> *****
> 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
>

[Lu Yan]

*****
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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:

> *****
> 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
>

[Alex Asanov]

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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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http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
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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

*****
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
>

---
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[Lu Yan]

*****
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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

*****
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
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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:

> *****
> 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
>

---
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[Rusty Nicovich]

*****
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*****

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.

Thanks,
Rusty

On Tue, Jun 5, 2018 at 11:27 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.
> *****
>
> 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
>

[Tala Allbutt]

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Hi Lu

It might be the methanol fixation. That could contribute to AF quite a bit. Also is the tissue from perfused animals? If not, could it be red blood cells/blood clots? They AF like crazy. Lipofuscin is a huge issue in imaging human brain but haven't come across as a problem in rodents (although perhaps aged rodents might be a different story). Lastly, although this may not be relevant here as you said the same dots were present in non-stained controls, too much fluorophore on the sample can often lead to bright dots like that.

Hope this may provide some leads for you to investigate.

Best wishes
Tala



Sent from myMail for iOS


Friday, June 1, 2018, 1:38 PM +1000 from [hidden email] <[hidden email]>:
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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

[Lu Yan]

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Hi Rusty,

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.

Thanks,
Lu

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Rusty Nicovich
Sent: Thursday, June 07, 2018 3:24 PM
To: [hidden email]
Subject: Re: Tissue imaging auto fluorescence and bright dot-like signal

*****
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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.

Thanks,
Rusty

On Tue, Jun 5, 2018 at 11:27 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.
> *****
>
> 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
>

[Lu Yan]

*****
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*****

Hi Tala,

Thanks much for the info. The tissue is from perfused mice. Regarding the dots in non-stained controls, we found clearing using pk and sds reduced the af a lot, e.g. the raw intensity dropped from 6~7k to ~1k whereas the probe signal can reach to 6k or so. But these dots are different under different channels (spatial distribution also differed). blue e.g. alexa 488, and green e.g. cy3 have the strongest signal, and they seem to be different dots under different channels.

Thanks,
Lu

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tamila Kaplinovsky
Sent: Thursday, June 07, 2018 4:02 PM
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
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*****

Hi Lu

It might be the methanol fixation. That could contribute to AF quite a bit. Also is the tissue from perfused animals? If not, could it be red blood cells/blood clots? They AF like crazy. Lipofuscin is a huge issue in imaging human brain but haven't come across as a problem in rodents (although perhaps aged rodents might be a different story). Lastly, although this may not be relevant here as you said the same dots were present in non-stained controls, too much fluorophore on the sample can often lead to bright dots like that.

Hope this may provide some leads for you to investigate.

Best wishes
Tala



Sent from myMail for iOS


Friday, June 1, 2018, 1:38 PM +1000 from [hidden email] <[hidden email]>:
*****
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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

[Glen MacDonald]

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How long are the perfused brains sitting in fixative or buffer before sectioning?  Formaldehyde fixation may reverse with prolonged storage and bits of tissue begin to come apart and absorb label.  Worse, bacterial contamination occurs which seems to either ingest  labeling components or the fluorophores stick to the bacterial glycocalyx.  This may be tested by observing mounted sections under a non-hardening mounting medium and running a short time-lapse to see whether your dots are moving.  

Glen

> On Jun 7, 2018, at 10:22 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.
> *****
>
> Hi Tala,
>
> Thanks much for the info. The tissue is from perfused mice. Regarding the dots in non-stained controls, we found clearing using pk and sds reduced the af a lot, e.g. the raw intensity dropped from 6~7k to ~1k whereas the probe signal can reach to 6k or so. But these dots are different under different channels (spatial distribution also differed). blue e.g. alexa 488, and green e.g. cy3 have the strongest signal, and they seem to be different dots under different channels.
>
> Thanks,
> Lu
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Tamila Kaplinovsky
> Sent: Thursday, June 07, 2018 4:02 PM
> 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.
> *****
>
> Hi Lu
>
> It might be the methanol fixation. That could contribute to AF quite a bit. Also is the tissue from perfused animals? If not, could it be red blood cells/blood clots? They AF like crazy. Lipofuscin is a huge issue in imaging human brain but haven't come across as a problem in rodents (although perhaps aged rodents might be a different story). Lastly, although this may not be relevant here as you said the same dots were present in non-stained controls, too much fluorophore on the sample can often lead to bright dots like that.
>
> Hope this may provide some leads for you to investigate.
>
> Best wishes
> Tala
>
>
>
> Sent from myMail for iOS
>
>
> Friday, June 1, 2018, 1:38 PM +1000 from [hidden email] <[hidden email]>:
> *****
> 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
>