Removing excess surface fluorophore on spinal column

[Craig Brideau]

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Hello listservians, I have a user working with cleared spinal column. He's
staining for lactase and overall it seems to be penetrating the tissue
well. The problem is his Alexa594 ends up accumulating in random material
on the surface of the tissue. When he images a volume, the very bright
signal at the top of the tissue tends to trip the PMTs leading to a fair
bit of frustration. Do any listers have recommendations for removing the
stain from the 'junk' on the surface of the sample? It is cleared tissue
which is about the consistency of jelly, so scraping would not be preferred.

Thanks,
Craig

[Rosemary.White]

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Hi Craig,

If this were plant tissue we'd dip it briefly in a moderate concentration of quenching agent, for a short enough time that this agent can't penetrate very far. If the quencher were rather large, that'd help keep it out of your stained tissue.

cheers,
Rosemary.
________________________________________
From: Confocal Microscopy List [[hidden email]] on behalf of Craig Brideau [[hidden email]]
Sent: Friday, 29 September 2017 7:37 a.m.
To: [hidden email]
Subject: Removing excess surface fluorophore on spinal column

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Hello listservians, I have a user working with cleared spinal column. He's
staining for lactase and overall it seems to be penetrating the tissue
well. The problem is his Alexa594 ends up accumulating in random material
on the surface of the tissue. When he images a volume, the very bright
signal at the top of the tissue tends to trip the PMTs leading to a fair
bit of frustration. Do any listers have recommendations for removing the
stain from the 'junk' on the surface of the sample? It is cleared tissue
which is about the consistency of jelly, so scraping would not be preferred.

Thanks,
Craig

[Craig Brideau]

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Sounds like a good idea. Can you suggest any quenchers for Alexa594?

Craig

On Thu, Sep 28, 2017 at 3:56 PM, <[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 Craig,
>
> If this were plant tissue we'd dip it briefly in a moderate concentration
> of quenching agent, for a short enough time that this agent can't penetrate
> very far. If the quencher were rather large, that'd help keep it out of
> your stained tissue.
>
> cheers,
> Rosemary.
> ________________________________________
> From: Confocal Microscopy List [[hidden email]] on
> behalf of Craig Brideau [[hidden email]]
> Sent: Friday, 29 September 2017 7:37 a.m.
> To: [hidden email]
> Subject: Removing excess surface fluorophore on spinal column
>
> *****
> 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.
> *****
>
> Hello listservians, I have a user working with cleared spinal column. He's
> staining for lactase and overall it seems to be penetrating the tissue
> well. The problem is his Alexa594 ends up accumulating in random material
> on the surface of the tissue. When he images a volume, the very bright
> signal at the top of the tissue tends to trip the PMTs leading to a fair
> bit of frustration. Do any listers have recommendations for removing the
> stain from the 'junk' on the surface of the sample? It is cleared tissue
> which is about the consistency of jelly, so scraping would not be
> preferred.
>
> Thanks,
> Craig
>

[Rosemary.White]

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Ah yes, that's the crunch!! As I pressed "send" the same thought went through my mind! But it'd be worth trying the ones that work on many dyes (for plants, at least) - toluidine blue, trypan blue, etc. Whoever now sells the Alexa dyes may have some hints.
cheers,
Rosemary

On 29/9/17, 8:14 am, "Confocal Microscopy List on behalf of Craig Brideau" <[hidden email] on behalf of [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.
    *****
   
    Sounds like a good idea. Can you suggest any quenchers for Alexa594?
   
    Craig
   
    On Thu, Sep 28, 2017 at 3:56 PM, <[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 Craig,
    >
    > If this were plant tissue we'd dip it briefly in a moderate concentration
    > of quenching agent, for a short enough time that this agent can't penetrate
    > very far. If the quencher were rather large, that'd help keep it out of
    > your stained tissue.
    >
    > cheers,
    > Rosemary.
    > ________________________________________
    > From: Confocal Microscopy List [[hidden email]] on
    > behalf of Craig Brideau [[hidden email]]
    > Sent: Friday, 29 September 2017 7:37 a.m.
    > To: [hidden email]
    > Subject: Removing excess surface fluorophore on spinal column
    >
    > *****
    > 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.
    > *****
    >
    > Hello listservians, I have a user working with cleared spinal column. He's
    > staining for lactase and overall it seems to be penetrating the tissue
    > well. The problem is his Alexa594 ends up accumulating in random material
    > on the surface of the tissue. When he images a volume, the very bright
    > signal at the top of the tissue tends to trip the PMTs leading to a fair
    > bit of frustration. Do any listers have recommendations for removing the
    > stain from the 'junk' on the surface of the sample? It is cleared tissue
    > which is about the consistency of jelly, so scraping would not be
    > preferred.
    >
    > Thanks,
    > Craig
    >
   

[Sathya Srinivasan]

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Hi Craig,
You can control the fluorophore intensity using the microscope settings (I
know you will find this option more attractive) and using pre-treatment
methods to quench the fluorophores.

Microscope settings to control intensity:
Most of the microscopes (including Nikon confocal- NIS ElementsC or AR
control software which I assume you are using, Leica it is called Z
compensation) come with a Z intensity correction control (In NIS Elements C
it is called Z intensity Control or Correction tab) where you can set the
intensity for different Z positions. The tab is under Capture Z-series
where you can see a button named "Z intensity control". We have done this
before while capturing clarified tissue using the A1R confocal at the RUN
but to gradually capture more signal as we image deeper into the tissue. In
your case this should be reversed, the first few Z planes should be set to
capture less signal by adjusting the parameters under the Acquisiton tab
and as you move into the tissue, you can set to capture optimal signal. If
you need a detailed step by step how to for the same, let me know and I can
send you.

Other methods to quench fluorophore intensity:
You can find a few articles about quenching fluorescence if you search
articles on troubleshooting autofluorescence. Most of the methods used
there are applicable to quench fluorescence too. You have to standardize
how long you have to expose the tissue to get the right amount of desired
fluorescence.

- M.S. Viegas, T.C. Martins, F. Seco, A. do Carmo. An improved and
cost-effective methodology for the reduction of autofluorescence in direct
immunofluorescence studies on formalin-fixed paraffin-embedded tissues.
European Journal of Histochemistry 2007; vol. 51 issue 1 (Jan-Mar): 59-66

- Michael Neumann and Detlef Gabel. Simple Method for Reduction of
Autofluorescence in Fluorescence Microscopy. J Histochem Cytochem 2002 50:
437

- V.C. Oliveira, et al. Sudan Black B treatment reduces autofluorescence
and improves resolution of in situ hybridization specific fluorescent
signals of brain sections. Histol Histopathol (2010) 25: 1017-1024.

If you need the PDFs, I can send you. Let me know and good luck.

Sathya Srinivasan
ONPRC



On Thu, Sep 28, 2017 at 3:59 PM, <[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.
> *****
>
> Ah yes, that's the crunch!! As I pressed "send" the same thought went
> through my mind! But it'd be worth trying the ones that work on many dyes
> (for plants, at least) - toluidine blue, trypan blue, etc. Whoever now
> sells the Alexa dyes may have some hints.
> cheers,
> Rosemary
>
> On 29/9/17, 8:14 am, "Confocal Microscopy List on behalf of Craig Brideau"
> <[hidden email] on behalf of [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.
>     *****
>
>     Sounds like a good idea. Can you suggest any quenchers for Alexa594?
>
>     Craig
>
>     On Thu, Sep 28, 2017 at 3:56 PM, <[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 Craig,
>     >
>     > If this were plant tissue we'd dip it briefly in a moderate
> concentration
>     > of quenching agent, for a short enough time that this agent can't
> penetrate
>     > very far. If the quencher were rather large, that'd help keep it out
> of
>     > your stained tissue.
>     >
>     > cheers,
>     > Rosemary.
>     > ________________________________________
>     > From: Confocal Microscopy List [[hidden email]] on
>     > behalf of Craig Brideau [[hidden email]]
>     > Sent: Friday, 29 September 2017 7:37 a.m.
>     > To: [hidden email]
>     > Subject: Removing excess surface fluorophore on spinal column
>     >
>     > *****
>     > 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.
>     > *****
>     >
>     > Hello listservians, I have a user working with cleared spinal
> column. He's
>     > staining for lactase and overall it seems to be penetrating the
> tissue
>     > well. The problem is his Alexa594 ends up accumulating in random
> material
>     > on the surface of the tissue. When he images a volume, the very
> bright
>     > signal at the top of the tissue tends to trip the PMTs leading to a
> fair
>     > bit of frustration. Do any listers have recommendations for removing
> the
>     > stain from the 'junk' on the surface of the sample? It is cleared
> tissue
>     > which is about the consistency of jelly, so scraping would not be
>     > preferred.
>     >
>     > Thanks,
>     > Craig
>     >
>
>
>

[Craig Brideau]

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On Fri, Sep 29, 2017 at 12:50 PM, Sathya Srinivasan <[hidden email]
> wrote:

> *****
> Microscope settings to control intensity:
> Most of the microscopes (including Nikon confocal- NIS ElementsC or AR
> control software which I assume you are using, Leica it is called Z
> compensation) come with a Z intensity correction control (In NIS Elements C
> it is called Z intensity Control or Correction tab) where you can set the
> intensity for different Z positions.


Yes, we are of course already doing that. The tricky part here is the
'coating' of Alexa594 is also down the *side* of our sample. Think of it as
the label on a tin can, where the can is the spinal column and the label is
the coating. We are imaging near the side of the 'can' so we pick up the
cross section of the 'label' regardless of depth. As an aside, we are also
using Z-intensity control to adjust power for depth already.

The tab is under Capture Z-series where you can see a button named "Z
> intensity control".


I am actually using a Bergamo 2, which allows you to input custom power
profiles for Z-stacks. It works quite well for normalizing signal over
significant depths. My user caught on to the interface very quickly and was
able to select an optimal exponential roll-off with Z right away.

Other methods to quench fluorophore intensity:
> You can find a few articles about quenching fluorescence if you search
> articles on troubleshooting autofluorescence. Most of the methods used
> there are applicable to quench fluorescence too. You have to standardize
> how long you have to expose the tissue to get the right amount of desired
> fluorescence.
>

Most of these articles address autofluorescence quenching, but my issue
here is not innate signal from the tissue. Instead I have an over
accumulation of fluorophore at the surface of a cleared tissue section
despite washing steps. Some users have suggested that there may be
something in the clearing or fixing process that may be trapping excess
secondary against the outside of the tissue. We've also confirmed that the
secondaries are raised in different animals and don't suspect spurious
antibody binding per se, it seems to be more of a mechanical pooling or
pocketing effect. We will need to either clean out the pools or
quench/bleach/destroy the Alexa594 in the accumulations. The most helpful
responses have suggested looking into an enzymatic or oxidative 'dip' of
the tissue to try to clean off that outer layer. I'm still exploring what
compounds would be safe to use (don't want it to penetrate into the tissue
much!) but I don't have any specific candidates so far.

Craig

>
>
> On Thu, Sep 28, 2017 at 3:59 PM, <[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.
> > *****
> >
> > Ah yes, that's the crunch!! As I pressed "send" the same thought went
> > through my mind! But it'd be worth trying the ones that work on many dyes
> > (for plants, at least) - toluidine blue, trypan blue, etc. Whoever now
> > sells the Alexa dyes may have some hints.
> > cheers,
> > Rosemary
> >
> > On 29/9/17, 8:14 am, "Confocal Microscopy List on behalf of Craig
> Brideau"
> > <[hidden email] on behalf of [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.
> >     *****
> >
> >     Sounds like a good idea. Can you suggest any quenchers for Alexa594?
> >
> >     Craig
> >
> >     On Thu, Sep 28, 2017 at 3:56 PM, <[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 Craig,
> >     >
> >     > If this were plant tissue we'd dip it briefly in a moderate
> > concentration
> >     > of quenching agent, for a short enough time that this agent can't
> > penetrate
> >     > very far. If the quencher were rather large, that'd help keep it
> out
> > of
> >     > your stained tissue.
> >     >
> >     > cheers,
> >     > Rosemary.
> >     > ________________________________________
> >     > From: Confocal Microscopy List [[hidden email]]
> on
> >     > behalf of Craig Brideau [[hidden email]]
> >     > Sent: Friday, 29 September 2017 7:37 a.m.
> >     > To: [hidden email]
> >     > Subject: Removing excess surface fluorophore on spinal column
> >     >
> >     > *****
> >     > 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.
> >     > *****
> >     >
> >     > Hello listservians, I have a user working with cleared spinal
> > column. He's
> >     > staining for lactase and overall it seems to be penetrating the
> > tissue
> >     > well. The problem is his Alexa594 ends up accumulating in random
> > material
> >     > on the surface of the tissue. When he images a volume, the very
> > bright
> >     > signal at the top of the tissue tends to trip the PMTs leading to a
> > fair
> >     > bit of frustration. Do any listers have recommendations for
> removing
> > the
> >     > stain from the 'junk' on the surface of the sample? It is cleared
> > tissue
> >     > which is about the consistency of jelly, so scraping would not be
> >     > preferred.
> >     >
> >     > Thanks,
> >     > Craig
> >     >
> >
> >
> >
>

[Konstantín Levitskiy]

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Hi Craig.

Could you think about the possibility first to put a competitive secondary antibody for a short time (for IR range), then wash, and after that putting the correct Alexa594 ?

Regards,
Dr. Konstantín Levitskiy
Servicio de Microscopía
InstitutodeBiomedicinadeSevilla - IBiS
Campus del Hospital Universitario Virgen del Rocío
Avda. Manuel Siurot s/nº
41013 Sevilla
Tlfno: 955 92 3030
Email: [hidden email]
Web: www.ibis-sevilla.es



-----Mensaje original-----
De: Confocal Microscopy List [mailto:[hidden email]] En nombre de Craig Brideau
Enviado el: jueves, 28 de septiembre de 2017 23:37
Para: [hidden email]
Asunto: Removing excess surface fluorophore on spinal column

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*****

Hello listservians, I have a user working with cleared spinal column. He's staining for lactase and overall it seems to be penetrating the tissue well. The problem is his Alexa594 ends up accumulating in random material on the surface of the tissue. When he images a volume, the very bright signal at the top of the tissue tends to trip the PMTs leading to a fair bit of frustration. Do any listers have recommendations for removing the stain from the 'junk' on the surface of the sample? It is cleared tissue which is about the consistency of jelly, so scraping would not be preferred.

Thanks,
Craig

[Craig Brideau]

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On Tue, Oct 3, 2017 at 5:46 AM, Konstantín Levitskiy <[hidden email]
> wrote:

> Could you think about the possibility first to put a competitive secondary
> antibody for a short time (for IR range), then wash, and after that putting
> the correct Alexa594 ?
>
> Hmmm... that would let us know whether it was truly binding to the
exterior, or just 'pooling' outside the tissue somehow. We'll see how the
other suggestions go and if we haven't ruled out actual epitope binding on
the surface we'll give this a try!

Craig

> Regards,
> Dr. Konstantín Levitskiy
> Servicio de Microscopía
> InstitutodeBiomedicinadeSevilla - IBiS
> Campus del Hospital Universitario Virgen del Rocío
> Avda. Manuel Siurot s/nº
> 41013 Sevilla
> Tlfno: 955 92 3030
> Email: [hidden email]
> Web: www.ibis-sevilla.es
>
>
>
> -----Mensaje original-----
> De: Confocal Microscopy List [mailto:[hidden email]] En
> nombre de Craig Brideau
> Enviado el: jueves, 28 de septiembre de 2017 23:37
> Para: [hidden email]
> Asunto: Removing excess surface fluorophore on spinal column
>
> *****
> 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.
> *****
>
> Hello listservians, I have a user working with cleared spinal column. He's
> staining for lactase and overall it seems to be penetrating the tissue
> well. The problem is his Alexa594 ends up accumulating in random material
> on the surface of the tissue. When he images a volume, the very bright
> signal at the top of the tissue tends to trip the PMTs leading to a fair
> bit of frustration. Do any listers have recommendations for removing the
> stain from the 'junk' on the surface of the sample? It is cleared tissue
> which is about the consistency of jelly, so scraping would not be preferred.
>
> Thanks,
> Craig
>

[Konstantín Levitskiy]

*****
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*****

Hi Craig.

1.

> or just 'pooling' outside the tissue somehow

This, I suppose, you (the user) could check, in part, testing without primary antibody. And for this, it's no need to clarify the tissue, I think.

Developing my first advice, it would be better for competitive step to add a mixture of secondary antibodies (desired Alexa594 and one in IR range [not occupied optical zone]) with different proportions in order to check (and to be able to choose) a decreased intensity of superficial Alexa594 staining. And again it's no need to clarifying the tissue, I suppose. You (the user) could have several transversal slices (of about 50-200 mkm) for studying different conditions for perimeter staining intensities.

2.
On the other hand, could you make a reflection acquisition on your confocal system? The idea is to subtract the reflection channel intensity acquisition from the fluorescence (594) one. Could clarified tissue have some reflection signal? I don't know. But you (the user) could try. It would be better to check several laser lines for doing that. And, I suppose the best result you will get with longest wavelength laser line. Remember the turbidity is measured using 680nm wavelength.

Good luck,
Konstantin

-----Mensaje original-----
De: Confocal Microscopy List [mailto:[hidden email]] En nombre de Craig Brideau
Enviado el: martes, 03 de octubre de 2017 19:26
Para: [hidden email]
Asunto: Re: Removing excess surface fluorophore on spinal column

On Tue, Oct 3, 2017 at 5:46 AM, Konstantín Levitskiy <[hidden email]
> wrote:

> Could you think about the possibility first to put a competitive
> secondary antibody for a short time (for IR range), then wash, and
> after that putting the correct Alexa594 ?
>
> Hmmm... that would let us know whether it was truly binding to the
exterior, or just 'pooling' outside the tissue somehow. We'll see how the other suggestions go and if we haven't ruled out actual epitope binding on the surface we'll give this a try!

Craig

> Regards,
> Dr. Konstantín Levitsky
> Servicio de Microscopía
> InstitutodeBiomedicinadeSevilla - IBiS Campus del Hospital
> Universitario Virgen del Rocío Avda. Manuel Siurot s/nº
> 41013 Sevilla
> Tlfno: 955 92 3030
> Email: [hidden email]
> Web: www.ibis-sevilla.es
>
> -----Mensaje original-----
> De: Confocal Microscopy List [mailto:[hidden email]]
> En nombre de Craig Brideau Enviado el: jueves, 28 de septiembre de
> 2017 23:37
> Para: [hidden email]
> Asunto: Removing excess surface fluorophore on spinal column
>
> Hello listservians, I have a user working with cleared spinal column.
> He's staining for lactase and overall it seems to be penetrating the
> tissue well. The problem is his Alexa594 ends up accumulating in
> random material on the surface of the tissue. When he images a volume,
> the very bright signal at the top of the tissue tends to trip the PMTs
> leading to a fair bit of frustration. Do any listers have
> recommendations for removing the stain from the 'junk' on the surface
> of the sample? It is cleared tissue which is about the consistency of jelly, so scraping would not be preferred.
>
> Thanks,
> Craig

[Craig Brideau]

*****
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On Wed, Oct 4, 2017 at 3:57 AM, Konstantín Levitskiy <[hidden email]
> wrote:

> > or just 'pooling' outside the tissue somehow
>
> This, I suppose, you (the user) could check, in part, testing without
> primary antibody. And for this, it's no need to clarify the tissue, I think.
>
> Developing my first advice, it would be better for competitive step to add
> a mixture of secondary antibodies (desired Alexa594 and one in IR range
> [not occupied optical zone]) with different proportions in order to check
> (and to be able to choose) a decreased intensity of superficial Alexa594
> staining. And again it's no need to clarifying the tissue, I suppose. You
> (the user) could have several transversal slices (of about 50-200 mkm) for
> studying different conditions for perimeter staining intensities.
>

I think I have some 647 lying around that might just work. I'll suggest it
to my user.


> On the other hand, could you make a reflection acquisition on your
> confocal system? The idea is to subtract the reflection channel intensity
> acquisition from the fluorescence (594) one. Could clarified tissue have
> some reflection signal? I don't know. But you (the user) could try. It
> would be better to check several laser lines for doing that. And, I suppose
> the best result you will get with longest wavelength laser line. Remember
> the turbidity is measured using 680nm wavelength.
>

Unfortunately it is a dedicated multiphoton system (Bergamo2) so no
confocal pinhole.

On another note:
I do have dual-path scanning with a slow galvo. One of my latest ideas
might be to selectively photobleach the bright spots using the slow galvos
while imaging with the fast galvos. I never imagined using them for
something like this but here we are. I just need to figure out how to
configure the scan paths but it should be pretty straightforward.

Craig

>
> Good luck,
> Konstantin
>
> -----Mensaje original-----
> De: Confocal Microscopy List [mailto:[hidden email]] En
> nombre de Craig Brideau
> Enviado el: martes, 03 de octubre de 2017 19:26
> Para: [hidden email]
> Asunto: Re: Removing excess surface fluorophore on spinal column
>
> On Tue, Oct 3, 2017 at 5:46 AM, Konstantín Levitskiy <
> [hidden email]
> > wrote:
>
> > Could you think about the possibility first to put a competitive
> > secondary antibody for a short time (for IR range), then wash, and
> > after that putting the correct Alexa594 ?
> >
> > Hmmm... that would let us know whether it was truly binding to the
> exterior, or just 'pooling' outside the tissue somehow. We'll see how the
> other suggestions go and if we haven't ruled out actual epitope binding on
> the surface we'll give this a try!
>
> Craig
>
> > Regards,
> > Dr. Konstantín Levitsky
> > Servicio de Microscopía
> > InstitutodeBiomedicinadeSevilla - IBiS Campus del Hospital
> > Universitario Virgen del Rocío Avda. Manuel Siurot s/nº
> > 41013 Sevilla
> > Tlfno: 955 92 3030
> > Email: [hidden email]
> > Web: www.ibis-sevilla.es
> >
> > -----Mensaje original-----
> > De: Confocal Microscopy List [mailto:[hidden email]]
> > En nombre de Craig Brideau Enviado el: jueves, 28 de septiembre de
> > 2017 23:37
> > Para: [hidden email]
> > Asunto: Removing excess surface fluorophore on spinal column
> >
> > Hello listservians, I have a user working with cleared spinal column.
> > He's staining for lactase and overall it seems to be penetrating the
> > tissue well. The problem is his Alexa594 ends up accumulating in
> > random material on the surface of the tissue. When he images a volume,
> > the very bright signal at the top of the tissue tends to trip the PMTs
> > leading to a fair bit of frustration. Do any listers have
> > recommendations for removing the stain from the 'junk' on the surface
> > of the sample? It is cleared tissue which is about the consistency of
> jelly, so scraping would not be preferred.
> >
> > Thanks,
> > Craig
>

[Konstantín Levitskiy]

*****
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*****

Hi Craig.

> I do have dual-path scanning with a slow galvo. One of my latest ideas might be to selectively photobleach the bright spots using the slow galvos while imaging with the fast galvos. I never imagined using them for something like this but here we are. I just need to figure out how to configure the scan paths but it should be pretty straightforward.

It's a good tricky idea. But, could you explain this for experimental protocol for Methods for an eventual publication? The method have to be clear to be reproducible for any other lab. It would be better to have some modification in the tissue preparation, that art-made adjustments during acquisition, wouldn't it? What do you think?

Best regards,
Konstantin

-----Mensaje original-----
De: Confocal Microscopy List [mailto:[hidden email]] En nombre de Craig Brideau
Enviado el: jueves, 05 de octubre de 2017 0:28
Para: [hidden email]
Asunto: Re: Removing excess surface fluorophore on spinal column

*****
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.
*****

On Wed, Oct 4, 2017 at 3:57 AM, Konstantín Levitskiy <[hidden email]
> wrote:

> > or just 'pooling' outside the tissue somehow
>
> This, I suppose, you (the user) could check, in part, testing without
> primary antibody. And for this, it's no need to clarify the tissue, I think.
>
> Developing my first advice, it would be better for competitive step to
> add a mixture of secondary antibodies (desired Alexa594 and one in IR
> range [not occupied optical zone]) with different proportions in order
> to check (and to be able to choose) a decreased intensity of
> superficial Alexa594 staining. And again it's no need to clarifying
> the tissue, I suppose. You (the user) could have several transversal
> slices (of about 50-200 mkm) for studying different conditions for perimeter staining intensities.
>

I think I have some 647 lying around that might just work. I'll suggest it to my user.


> On the other hand, could you make a reflection acquisition on your
> confocal system? The idea is to subtract the reflection channel
> intensity acquisition from the fluorescence (594) one. Could clarified
> tissue have some reflection signal? I don't know. But you (the user)
> could try. It would be better to check several laser lines for doing
> that. And, I suppose the best result you will get with longest
> wavelength laser line. Remember the turbidity is measured using 680nm wavelength.
>

Unfortunately it is a dedicated multiphoton system (Bergamo2) so no confocal pinhole.

On another note:
I do have dual-path scanning with a slow galvo. One of my latest ideas might be to selectively photobleach the bright spots using the slow galvos while imaging with the fast galvos. I never imagined using them for something like this but here we are. I just need to figure out how to configure the scan paths but it should be pretty straightforward.

Craig

>
> Good luck,
> Konstantin
>
> -----Mensaje original-----
> De: Confocal Microscopy List [mailto:[hidden email]]
> En nombre de Craig Brideau Enviado el: martes, 03 de octubre de 2017
> 19:26
> Para: [hidden email]
> Asunto: Re: Removing excess surface fluorophore on spinal column
>
> On Tue, Oct 3, 2017 at 5:46 AM, Konstantín Levitskiy <
> [hidden email]
> > wrote:
>
> > Could you think about the possibility first to put a competitive
> > secondary antibody for a short time (for IR range), then wash, and
> > after that putting the correct Alexa594 ?
> >
> > Hmmm... that would let us know whether it was truly binding to the
> exterior, or just 'pooling' outside the tissue somehow. We'll see how
> the other suggestions go and if we haven't ruled out actual epitope
> binding on the surface we'll give this a try!
>
> Craig
>
> > Regards,
> > Dr. Konstantín Levitsky
> > Servicio de Microscopía
> > InstitutodeBiomedicinadeSevilla - IBiS Campus del Hospital
> > Universitario Virgen del Rocío Avda. Manuel Siurot s/nº
> > 41013 Sevilla
> > Tlfno: 955 92 3030
> > Email: [hidden email]
> > Web: www.ibis-sevilla.es
> >
> > -----Mensaje original-----
> > De: Confocal Microscopy List
> > [mailto:[hidden email]]
> > En nombre de Craig Brideau Enviado el: jueves, 28 de septiembre de
> > 2017 23:37
> > Para: [hidden email]
> > Asunto: Removing excess surface fluorophore on spinal column
> >
> > Hello listservians, I have a user working with cleared spinal column.
> > He's staining for lactase and overall it seems to be penetrating the
> > tissue well. The problem is his Alexa594 ends up accumulating in
> > random material on the surface of the tissue. When he images a
> > volume, the very bright signal at the top of the tissue tends to
> > trip the PMTs leading to a fair bit of frustration. Do any listers
> > have recommendations for removing the stain from the 'junk' on the
> > surface of the sample? It is cleared tissue which is about the
> > consistency of
> jelly, so scraping would not be preferred.
> >
> > Thanks,
> > Craig
>