Experience with liver tissue autofluorescence ?

[Jean-Pierre CLAMME-2]

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Hi,

I'm looking for someone having experience with confocal imaging of liver
tissue. I'm imaging mouse liver tissue and I have some issues with
autofluorescence in the cy3 channel coming from structure appearing like
vesicle in hepatocytes. Those vesicles mainly show up in the Cy3 channel
but are also visible in the FITC channel (depending on the power I use).
A spectral image with excitation at 488, shows a broad signal with a
maximum around 580 nm.
Could someone comment on the origin of this fluorescence ? Is it possible
that it is lipofuscine ?

Thank you,

JP

[mcammer]

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Most likely lipid droplets.  

_________________________________________
Michael Cammer, Assistant Research Scientist
Skirball Institute of Biomolecular Medicine
Lab: [phone]  Cell: [phone]

________________________________________
From: Confocal Microscopy List [[hidden email]] On Behalf Of Jean-Pierre CLAMME [[hidden email]]
Sent: Tuesday, April 05, 2011 9:06 PM
To: [hidden email]
Subject: Experience with liver tissue autofluorescence ?

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Hi,

I'm looking for someone having experience with confocal imaging of liver
tissue. I'm imaging mouse liver tissue and I have some issues with
autofluorescence in the cy3 channel coming from structure appearing like
vesicle in hepatocytes. Those vesicles mainly show up in the Cy3 channel
but are also visible in the FITC channel (depending on the power I use).
A spectral image with excitation at 488, shows a broad signal with a
maximum around 580 nm.
Could someone comment on the origin of this fluorescence ? Is it possible
that it is lipofuscine ?

Thank you,

JP

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[Lemasters, John J.]

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Lipid droplets in stellate (Ito) cells are highly autofluorescent due to their content of esters of retinoic acid. These are probably your structures.

--
John J. Lemasters, MD, PhD
Professor and GlaxoSmithKline Distinguished Endowed Chair
Director, Center for Cell Death, Injury and Regeneration
Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology
Medical University of South Carolina
QF213 Quadrangle Building
280 Calhoun Street, MSC 140
Charleston, SC 29425

Office: 843-792-2153
Lab: 843-792-3530
Fax: [phone]
Email: [hidden email]
http://academicdepartments.musc.edu/ccdir


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Jean-Pierre CLAMME
Sent: Tuesday, April 05, 2011 9:06 PM
To: [hidden email]
Subject: Experience with liver tissue autofluorescence ?

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Hi,

I'm looking for someone having experience with confocal imaging of liver tissue. I'm imaging mouse liver tissue and I have some issues with autofluorescence in the cy3 channel coming from structure appearing like vesicle in hepatocytes. Those vesicles mainly show up in the Cy3 channel but are also visible in the FITC channel (depending on the power I use).
A spectral image with excitation at 488, shows a broad signal with a maximum around 580 nm.
Could someone comment on the origin of this fluorescence ? Is it possible that it is lipofuscine ?

Thank you,

JP

[Martin Wessendorf-2]

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Dear JP--

 From your description, it might be lipofuscin.  In my limited
experience, mouse tissue has a lot of it.  If that's what it is, it will
appear yellow/orange with a wide-band UV filter, green through a
fluorescein filter, red through a rhodamine filter and will also be
visible through a Cy5 filter.  It will be very resistant to
photobleaching.  It will remain fluorescent after treatment with NaBH4
or oxidizing agents or after lipid extraction.

You can deal with lipofuscin one of at least 3 ways:

1)  Don't use fluorescence--use immunoperoxidase methods.

2)  Treat with Cu++.  Cu++ ion will quench much of the autofluorescence
of lipofuscin, but it will also somewhat reduce the fluorescence of the
Cy3.

3)  Treat with Sudan Black.  Sudan Black binds lipophilic compartments
and, being black, quenches lipofuscin.  It's not compatible with
xylene-based mounting media, though.

Steve Schnell, Bill Staines and I have a paper on this:
Schnell SA, Staines WA, Wessendorf MW.  Reduction of lipofuscin-like
autofluorescence in fluorescently labeled tissue.  J Histochem Cytochem.
1999 47(6):719-30.

Good luck!

Martin Wessendorf

  On 4/5/2011 8:06 PM, Jean-Pierre CLAMME wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi,
>
> I'm looking for someone having experience with confocal imaging of liver
> tissue. I'm imaging mouse liver tissue and I have some issues with
> autofluorescence in the cy3 channel coming from structure appearing like
> vesicle in hepatocytes. Those vesicles mainly show up in the Cy3 channel
> but are also visible in the FITC channel (depending on the power I use).
> A spectral image with excitation at 488, shows a broad signal with a
> maximum around 580 nm.
> Could someone comment on the origin of this fluorescence ? Is it possible
> that it is lipofuscine ?
>
> Thank you,
>
> JP

--
Martin Wessendorf, Ph.D.                   office: [phone]
Assoc Prof, Dept Neuroscience                 lab: [phone]
University of Minnesota             Preferred FAX: [phone]
6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
Minneapolis, MN  55455                    e-mail: [hidden email]

[Jean-Pierre CLAMME-2]

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Based on their spectra, I wouldn't expect the fluorescence of esters of
retinoids to be fluorescent in the Cy3 channel  but more in the DAPI
channel. Also I mainly see those structures around the round nucleus of
hepatocytes.


> Lipid droplets in stellate (Ito) cells are highly autofluorescent
> due to their content of esters of retinoic acid. These are probably
> your structures.

> --
> John J. Lemasters, MD, PhD
> Professor and GlaxoSmithKline Distinguished Endowed Chair
> Director, Center for Cell Death, Injury and Regeneration
> Departments of Pharmaceutical & Biomedical Sciences and Biochemistry
> & Molecular Biology
> Medical University of South Carolina
> QF213 Quadrangle Building
> 280 Calhoun Street, MSC 140
> Charleston, SC 29425

> Office: 843-792-2153
> Lab: 843-792-3530
> Fax: [phone]
> Email: [hidden email]
> http://academicdepartments.musc.edu/ccdir

>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email].
> EDU] On Behalf Of Jean-Pierre CLAMME
> Sent: Tuesday, April 05, 2011 9:06 PM
> To: [hidden email]
> Subject: Experience with liver tissue autofluorescence ?

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****

> Hi,

> I'm looking for someone having experience with confocal imaging of
> liver tissue. I'm imaging mouse liver tissue and I have some issues
> with autofluorescence in the cy3 channel coming from structure
> appearing like vesicle in hepatocytes. Those vesicles mainly show up
> in the Cy3 channel but are also visible in the FITC channel
> (depending on the power I use).
> A spectral image with excitation at 488, shows a broad signal with a
> maximum around 580 nm.
> Could someone comment on the origin of this fluorescence ? Is it
> possible that it is lipofuscine ?

> Thank you,

> JP

[Jean-Pierre CLAMME-2]

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Thank you very much !



Confocal Microscopy List <[hidden email]> wrote on
04/05/2011 06:35:56 PM:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****

> Dear JP--

> From your description, it might be lipofuscin.  In my limited
> experience, mouse tissue has a lot of it.  If that's what it is, it will
> appear yellow/orange with a wide-band UV filter, green through a
> fluorescein filter, red through a rhodamine filter and will also be
> visible through a Cy5 filter.  It will be very resistant to
> photobleaching.  It will remain fluorescent after treatment with NaBH4
> or oxidizing agents or after lipid extraction.

> You can deal with lipofuscin one of at least 3 ways:

> 1)  Don't use fluorescence--use immunoperoxidase methods.

> 2)  Treat with Cu++.  Cu++ ion will quench much of the autofluorescence
> of lipofuscin, but it will also somewhat reduce the fluorescence of the
> Cy3.

> 3)  Treat with Sudan Black.  Sudan Black binds lipophilic compartments
> and, being black, quenches lipofuscin.  It's not compatible with
> xylene-based mounting media, though.

> Steve Schnell, Bill Staines and I have a paper on this:
> Schnell SA, Staines WA, Wessendorf MW.  Reduction of lipofuscin-like
> autofluorescence in fluorescently labeled tissue.  J Histochem Cytochem.
> 1999 47(6):719-30.

> Good luck!

> Martin Wessendorf

> On 4/5/2011 8:06 PM, Jean-Pierre CLAMME wrote:
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi,
> >
> > I'm looking for someone having experience with confocal imaging of
liver
> > tissue. I'm imaging mouse liver tissue and I have some issues with
> > autofluorescence in the cy3 channel coming from structure appearing
like
> > vesicle in hepatocytes. Those vesicles mainly show up in the Cy3
channel
> > but are also visible in the FITC channel (depending on the power I
use).
> > A spectral image with excitation at 488, shows a broad signal with a
> > maximum around 580 nm.
> > Could someone comment on the origin of this fluorescence ? Is it
possible
> > that it is lipofuscine ?
> >
> > Thank you,
> >
> > JP

>
> --
> Martin Wessendorf, Ph.D.                   office: [phone]
> Assoc Prof, Dept Neuroscience                 lab: [phone]
> University of Minnesota             Preferred FAX: [phone]
> 6-145 Jackson Hall, 321 Church St. SE    Dept Fax: [phone]
> Minneapolis, MN  55455                    e-mail: [hidden email]