Subnuclear localization of transcription factors in fixed tissue

[Ella Tour]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Confocalists,

I would greatly appreciate your advice on the topic I don't know much about:
I am imaging several proteins belonging to a Hox group of transcription factors, which regulate multiple (hundreds) of genes by specifically binding DNA on single and clustered sites. I visualize these proteins using indirect immunofluorescence (primary polyclonal and secondary antibodies fused to Alexa dyes) in fixed Drosophila embryos and image them on Leica SP2 confocal microscope At high magnification, 63X and higher, these transcription factors appear as foci, or speckles, within the nucleus. I observe this speckled localization with a variety of antibodies against several transcription factors. After deconvolution using Autoquant software, these foci become even more profound, about 60 of them per nucleus.
Now here are my questions:
Are these foci real or an artifact (of fixation, for example)?

If they are real, are they just random clusters of proteins inside the nucleus or do I actually observe clusters of transcription factors bound to their DNA target sequences?

Does anybody have an estimate of how many proteins need to be clustered together in order to be detected with polyclonal antibodies/secondary antibodies with 5-6 fluorophores each, in fixed tissue (about 10-20 microns under the coverslip)?

I realize that some of these questions will probably not have general answers ( that is, the answers will vary depending which transcription factor and which system one is working with), but any input from people who image transcription factors or any referrals to the relevant literature will be greatly appreciated. 

Best wishes,

Ella Tour

-- 

Ella Tour, Ph.D.
Department of Cell and Developmental Biology, 0349                     
University of California, San Diego            
9500 Gilman Drive, 4305 Bonner Hall                            
La Jolla, CA  92093-0349
Phone [phone]
FAX [phone]
email: [hidden email]

[JOEL B. SHEFFIELD]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

There is, I suppose you know, a fair literature about "Speckles",
including some impressive studies of dynamic behavior of specific
protein components.  You might take a look at the work by Platani et
al. (Jcb. 151:1561-1574), or Sleeman, or Swedlow.  Much of this work
was done in 2000-2003.  There is a recent paper as well: PNAS October
3, 2006 vol. 103 _ no. 40 , about the "FLASH" protein.  Most of these
papers refer to "Cajal" bodies, but there are other components of
these speckles as well.  There is a nice review by Joe Gall in Annual
Review of Cell Biology, 2000.

I don't have an answer to your more general question about how many
antibodies are necessary for visualization of a protein aggregate --
An interesting question.  I hope that you share any answers you get.

Joel

> Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-
> bin/wa?S1=confocal
> Dear Confocalists,
>
> I would greatly appreciate your advice on the topic I don't know much
> about:
> I am imaging several proteins belonging to a Hox group of
> transcription factors, which regulate multiple (hundreds) of genes by
> specifically binding DNA on single and clustered sites. I visualize
> these proteins using indirect immunofluorescence (primary polyclonal
> and secondary antibodies fused to Alexa dyes) in fixed Drosophila
> embryos and image them on Leica SP2 confocal microscope At high
> magnification, 63X and higher, these transcription factors appear as
> foci, or speckles, within the nucleus. I observe this speckled
> localization with a variety of antibodies against several
> transcription factors. After deconvolution using Autoquant software,
> these foci become even more profound, about 60 of them per nucleus.
> Now here are my questions:
> Are these foci real or an artifact (of fixation, for example)?
>
> If they are real, are they just random clusters of proteins inside
> the nucleus or do I actually observe clusters of transcription
> factors bound to their DNA target sequences?
>
> Does anybody have an estimate of how many proteins need to be
> clustered together in order to be detected with polyclonal
> antibodies/secondary antibodies with 5-6 fluorophores each, in fixed
> tissue (about 10-20 microns under the coverslip)?
> I realize that some of these questions will probably not have general
> answers ( that is, the answers will vary depending which
> transcription factor and which system one is working with), but any
> input from people who image transcription factors or any referrals to
> the relevant literature will be greatly appreciated.
>
> Best wishes,
>
> Ella Tour
>
> --
>
>
>
>
>
> Ella Tour, Ph.D.
> Department of Cell and Developmental Biology, 0349  
> University of California, San Diego  
> 9500 Gilman Drive, 4305 Bonner Hall  
> La Jolla, CA 92093-0349
> Phone [phone]
> FAX [phone]
> email: [hidden email]

--
Joel B. Sheffield, Ph.D.
Biology Department, Temple University
1900 North 12th Street
Philadelphia, PA 19122
[hidden email]  
[phone], fax [phone]
http://astro.temple.edu/~jbs

[Farid Jalali]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hello Ella,
I am looking at sub-nuclear features as well, proteins that are phosphorylated at or near sites of DNA damage, or re-localize to/near sites of DNA damage. Distinguishing damage foci from background is a challenge. A couple of ideas that you may want to consider are dose response and kinetics; is there a change in appearance/numbers of speckles as a function of time or stimulus? This may help you determine what are the true foci vs. background; if they are dynamic in their formation/resolution then I would guess that they are not background. I would expect background to be non-dynamic. We try to use inhibitors of the targets we are interested in as controls for focus formation and resolution, or siRNA.

Sorry to not be able to provide you with more info. Good luck.
Farid

On Thu, Mar 20, 2008 at 8:03 PM, Ella Tour <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Confocalists,

I would greatly appreciate your advice on the topic I don't know much about:
I am imaging several proteins belonging to a Hox group of transcription factors, which regulate multiple (hundreds) of genes by specifically binding DNA on single and clustered sites. I visualize these proteins using indirect immunofluorescence (primary polyclonal and secondary antibodies fused to Alexa dyes) in fixed Drosophila embryos and image them on Leica SP2 confocal microscope At high magnification, 63X and higher, these transcription factors appear as foci, or speckles, within the nucleus. I observe this speckled localization with a variety of antibodies against several transcription factors. After deconvolution using Autoquant software, these foci become even more profound, about 60 of them per nucleus.
Now here are my questions:
Are these foci real or an artifact (of fixation, for example)?

If they are real, are they just random clusters of proteins inside the nucleus or do I actually observe clusters of transcription factors bound to their DNA target sequences?

Does anybody have an estimate of how many proteins need to be clustered together in order to be detected with polyclonal antibodies/secondary antibodies with 5-6 fluorophores each, in fixed tissue (about 10-20 microns under the coverslip)?

I realize that some of these questions will probably not have general answers ( that is, the answers will vary depending which transcription factor and which system one is working with), but any input from people who image transcription factors or any referrals to the relevant literature will be greatly appreciated. 

Best wishes,

Ella Tour

-- 

Ella Tour, Ph.D.
Department of Cell and Developmental Biology, 0349                     
University of California, San Diego            
9500 Gilman Drive, 4305 Bonner Hall                            
La Jolla, CA  92093-0349
Phone [phone]
FAX [phone]
email: [hidden email]

--
Farid Jalali MSc
Senior Research Technician/ Lab Manager
Dr. Robert Bristow Lab
Applied Molecular Oncology
Princess Margaret Hospital
Toronto, Canada
416-946-4501 X4351 (Princess Margaret Hospital)
416-581-7754 STTARR at MaRS Building
416-581-7791 STTARR Microscopy Suite

[Gert van Cappellen]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

It's off course hard to judge if your foci are real or artefact. For
many systems the formation of foci is known. In a recent paper of  van
Royen et al ( The Journal of Cell Biology, Vol. 177, No. 1, 63-72; 2007)
a relation between the Androgen receptor and active transcription was
shown by using, 5-bromo-uridine-5'-triphosphate^ (BrUTP). You might also
try to do this. The number of molecules in a focus will probably be
something between 20 and 500 molecules.

Best regards,
Gert van Cappellen

Ella Tour schreef:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
> Dear Confocalists,
>
> I would greatly appreciate your advice on the topic I don't know much
> about:
> I am imaging several proteins belonging to a Hox group of
> transcription factors, which regulate multiple (hundreds) of genes by
> specifically binding DNA on single and clustered sites. I visualize
> these proteins using indirect immunofluorescence (primary polyclonal
> and secondary antibodies fused to Alexa dyes) in fixed Drosophila
> embryos and image them on Leica SP2 confocal microscope At high
> magnification, 63X and higher, these transcription factors appear as
> foci, or speckles, within the nucleus. I observe this speckled
> localization with a variety of antibodies against several
> transcription factors. After deconvolution using Autoquant software,
> these foci become even more profound, about 60 of them per nucleus.
> Now here are my questions:
> Are these foci real or an artifact (of fixation, for example)?
>
> If they are real, are they just random clusters of proteins inside the
> nucleus or do I actually observe clusters of transcription factors
> bound to their DNA target sequences?
>
> Does anybody have an estimate of how many proteins need to be
> clustered together in order to be detected with polyclonal
> antibodies/secondary antibodies with 5-6 fluorophores each, in fixed
> tissue (about 10-20 microns under the coverslip)?
> I realize that some of these questions will probably not have general
> answers ( that is, the answers will vary depending which transcription
> factor and which system one is working with), but any input from
> people who image transcription factors or any referrals to the
> relevant literature will be greatly appreciated.
>
> Best wishes,
>
> Ella Tour
>
> --
>  
>
>
>
>
> Ella Tour, Ph.D.
> Department of Cell and Developmental Biology, 0349                    
> University of California, San Diego            
> 9500 Gilman Drive, 4305 Bonner Hall                            
> La Jolla, CA  92093-0349
> Phone [phone]
> FAX [phone]
> email: [hidden email]

[Jason Swedlow]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Ella-

There are various nuclear bodies, and they go by a variety of names.  David Spector & Angus Lamond have a nice discussion of speckles:

http://www.ncbi.nlm.nih.gov/pubmed/12923522

In addition, David Spector has a nice survey of many of the different nuclear domains:

http://www.ncbi.nlm.nih.gov/pubmed/11686292

Your questions:

1. Are these foci real?  Possibly.  There certainly are nuclear foci.  Best thing to do is compare your distribution by multi-channel immunofluorescence with markers for the various domains.  See the above papers for suggestions.

2. What are the clusters?  They can be many things-- storage sites for factors, processing sites, etc.  There may even be sites of action.  It's pretty clear that the various distributions one sees are many things, and not just sites of action.  This is covered in the papers above.

3. How many molecules are there?  Very hard to know-- depends on alot of things, including your sample prep, antibody, mounting, microscopy method, etc.  Quantifying immunofluorescence images is tricky.  For instance,

http://www.ncbi.nlm.nih.gov/pubmed/1527176

I suspect this list could start a long thread on this issue ;-)!

Hope that helps.

Cheers,

Jason

On Fri, Mar 21, 2008 at 12:03 AM, Ella Tour <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Confocalists,

I would greatly appreciate your advice on the topic I don't know much about:
I am imaging several proteins belonging to a Hox group of transcription factors, which regulate multiple (hundreds) of genes by specifically binding DNA on single and clustered sites. I visualize these proteins using indirect immunofluorescence (primary polyclonal and secondary antibodies fused to Alexa dyes) in fixed Drosophila embryos and image them on Leica SP2 confocal microscope At high magnification, 63X and higher, these transcription factors appear as foci, or speckles, within the nucleus. I observe this speckled localization with a variety of antibodies against several transcription factors. After deconvolution using Autoquant software, these foci become even more profound, about 60 of them per nucleus.
Now here are my questions:
Are these foci real or an artifact (of fixation, for example)?

If they are real, are they just random clusters of proteins inside the nucleus or do I actually observe clusters of transcription factors bound to their DNA target sequences?

Does anybody have an estimate of how many proteins need to be clustered together in order to be detected with polyclonal antibodies/secondary antibodies with 5-6 fluorophores each, in fixed tissue (about 10-20 microns under the coverslip)?

I realize that some of these questions will probably not have general answers ( that is, the answers will vary depending which transcription factor and which system one is working with), but any input from people who image transcription factors or any referrals to the relevant literature will be greatly appreciated. 

Best wishes,

Ella Tour

-- 

Ella Tour, Ph.D.
Department of Cell and Developmental Biology, 0349                     
University of California, San Diego            
9500 Gilman Drive, 4305 Bonner Hall                            
La Jolla, CA  92093-0349
Phone [phone]
FAX [phone]
email: [hidden email]

--
**************************
Wellcome Trust Centre for Gene Regulation & Expression
College of Life Sciences
MSI/WTB/JBC Complex
University of Dundee
Dow Street
Dundee DD1 5EH
United Kingdom

phone [phone]
Intl phone: [phone]
FAX [phone]
email: [hidden email]

Lab Page: http://www.dundee.ac.uk/lifesciences/swedlow/
Open Microscopy Environment: http://openmicroscopy.org
**************************

[Mancini, Michael A]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello Ella,

....a great microscopy topic, indeed, one of my favorites.

without seeing the images it is (essentially) impossible to say, but as Jason mentioned, there are lots of possibilities.  we've looked at transcription factor localization and dynamics for a while and have seen all sorts of speckles, blobs, foci, inclusions, aggregates, etc.  absolutely all of them are not physiologically relevant.  the most suspicious ones are any found after over-expression studies.  while the mainstay of most transcription factor studies, transient (or even some stable) transfections can be difficult to interpret by imaging-----unless you can clearly define what is considered an expression level that is close to what is observed when endogenously expressed.  i've seen a awful lot of 'interesting' patterns that are all too often the result of a weird misfolded protein (from a deletion or point mutation), or just something that sets the cell into a cell stress/toxic response.  

if you're seeing fine speckles throughout the nucleoplasm using a 'proven' antibody and in cells with endogenous expression, they're probably 'real.'  however, that does not mean they are transcription sites by any means.  most studies show pol II or BrUTP incorporation with such speckles as being at best partially colocalizing, and in many cases, are merely random in their association.  given that most of these TFs, when examined by GFP-fusion photobleaching, are markedly dynamic, the foci, in my opinion, are much more likely the reflection of transient complex formation off the specific target promoters, which are very rare (at least in terms of 3D geometry in the nucleus; e.g., the vast majority of 3D space is not transcriptional active).  Are they intentional "storage" or just  the stochastic result of molecular dynamics?   that's still a very difficult question to answer........  I do know one thing, it is very difficult to reconcile biochemical approaches like chip with localization studies-----they're very different beasts, each with their own pros and cons. 

best of luck with your studies,

Mike Mancini

Baylor College of Medicine

Houston, TX

On Mar 21, 2008, at 4:25 PM, Jason Swedlow wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi Ella-

There are various nuclear bodies, and they go by a variety of names.  David Spector & Angus Lamond have a nice discussion of speckles:

http://www.ncbi.nlm.nih.gov/pubmed/12923522

In addition, David Spector has a nice survey of many of the different nuclear domains:

http://www.ncbi.nlm.nih.gov/pubmed/11686292

Your questions:

1. Are these foci real?  Possibly.  There certainly are nuclear foci.  Best thing to do is compare your distribution by multi-channel immunofluorescence with markers for the various domains.  See the above papers for suggestions.

2. What are the clusters?  They can be many things-- storage sites for factors, processing sites, etc.  There may even be sites of action.  It's pretty clear that the various distributions one sees are many things, and not just sites of action.  This is covered in the papers above.

3. How many molecules are there?  Very hard to know-- depends on alot of things, including your sample prep, antibody, mounting, microscopy method, etc.  Quantifying immunofluorescence images is tricky.  For instance,

http://www.ncbi.nlm.nih.gov/pubmed/1527176

I suspect this list could start a long thread on this issue ;-)!

Hope that helps.

Cheers,

Jason

On Fri, Mar 21, 2008 at 12:03 AM, Ella Tour <[hidden email]> wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Confocalists,

I would greatly appreciate your advice on the topic I don't know much about:
I am imaging several proteins belonging to a Hox group of transcription factors, which regulate multiple (hundreds) of genes by specifically binding DNA on single and clustered sites. I visualize these proteins using indirect immunofluorescence (primary polyclonal and secondary antibodies fused to Alexa dyes) in fixed Drosophila embryos and image them on Leica SP2 confocal microscope At high magnification, 63X and higher, these transcription factors appear as foci, or speckles, within the nucleus. I observe this speckled localization with a variety of antibodies against several transcription factors. After deconvolution using Autoquant software, these foci become even more profound, about 60 of them per nucleus.
Now here are my questions:
Are these foci real or an artifact (of fixation, for example)?

If they are real, are they just random clusters of proteins inside the nucleus or do I actually observe clusters of transcription factors bound to their DNA target sequences?

Does anybody have an estimate of how many proteins need to be clustered together in order to be detected with polyclonal antibodies/secondary antibodies with 5-6 fluorophores each, in fixed tissue (about 10-20 microns under the coverslip)?

I realize that some of these questions will probably not have general answers ( that is, the answers will vary depending which transcription factor and which system one is working with), but any input from people who image transcription factors or any referrals to the relevant literature will be greatly appreciated. 

Best wishes,

Ella Tour

-- 

Ella Tour, Ph.D.
Department of Cell and Developmental Biology, 0349                     
University of California, San Diego            
9500 Gilman Drive, 4305 Bonner Hall                            
La Jolla, CA  92093-0349
Phone [phone]
FAX [phone]
email: [hidden email]

--
**************************
Wellcome Trust Centre for Gene Regulation & Expression
College of Life Sciences
MSI/WTB/JBC Complex
University of Dundee
Dow Street
Dundee DD1 5EH
United Kingdom

phone [phone]
Intl phone: [phone]
FAX [phone]
email: [hidden email]

Lab Page: http://www.dundee.ac.uk/lifesciences/swedlow/
Open Microscopy Environment: http://openmicroscopy.org
**************************

[Ramshesh, Venkat K]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Second Charleston Workshop on

LIGHT MICROSCOPY FOR THE BIOSCIENCES (LMB)

Medical University of South Carolina

May 18-23, 2008

The Second Charleston Workshop on LIGHT MICROSCOPY FOR THE BIOSCIENCES
(LMB) Workshop provides a solid introduction to the concepts and
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digital image processing, and fluorescence and confocal/multiphoton
microscopy guided by experienced academic and commercial faculty.
Lectures and laboratory exercises will include: optics of image
formation; microscope alignment; phase contrast and differential
interference contrast microscopy; video and digital cameras; contrast
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probes, including green fluorescent protein; fluorescence resonance
energy transfer; and laser scanning confocal and multiphoton microscopy.
A commercial faculty representing leading microscope manufacturers will
make available for student use the latest and most advanced
instrumentation for light microscopy, image detection and computerized
image analysis. The LMB Workshop is designed for doctoral level
scientists, advanced pre-doctoral students and high level technical
personnel. No prior experience with microscopy is required. All students
will benefit from in-depth interaction with instructors. Students are
encouraged to bring their own specimens for analysis.

Tuition: $750.00

Application Deadline: April 15, 2008

Principal Instructors:

John J. Lemasters, M.D., Ph.D., Organizer

P. Darwin Bell, Ph.D.

Margaret Kelly, Ph.D.

Peter Komlosi, Ph.D.

Anna-Liisa Nieminen, Ph.D.

Venkat Ramshesh, Ph.D.

To apply, send a curriculum vita and a brief letter describing your
research interests and reasons for enrolling. Because the course is
expected to be oversubscribed, applicants should inquire as soon as
possible. Please indicate your complete mailing address, telephone/fax
number and email address. Full consideration will be given to
applications received by April 15, 2008.

For further information or to apply, contact:

Venkat K Ramshesh
Medical University of South Carolina
Center for Cell Death, Injury and Regeneration and Hollings Cancer Center
280 Calhoun Street, PO Box 250140
Charleston, SC 29425
Telephone [phone], FAX [phone]
E-mail: [hidden email]

[Ella Tour]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Confocalists,

Thanks a lot for your useful suggestions and the plenitude of great references. I am still digging through them.

I will be definitely testing whether my protein foci colocalize with the markers for the known subnuclear structures, such as  the "speckles" (or "interchromatin granule clusters" in EM), Cajal  bodies and PML bodies.

 
Some of you have raised a very valid point: that protein aggregates might be a result of an over-expression of a protein at abnormally high levels. This is not the case in our system: most of the times, we detect the endogenous protein in embryos. The antibodies we work with are specific, as they detect the Hox proteins in the regions where their genes are transcribed.

Thank you very much again,

Ella

-- 

Ella Tour
Department of Cell and Developmental Biology, 0349                    
University of California, San Diego            
9500 Gilman Drive, 4305 Bonner Hall                            
La Jolla, CA  92093-0349
Phone [phone]
FAX [phone]
email: [hidden email]

[Boswell, Carl A - (cboswell)]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Ella,

One thing I didn't see mentioned in this thread was the possibility of aggregates or precipitates forming during fixation/denaturation.  It's the first thing I think of when I see the term "speckles" to describe a staining pattern.  A classic artifact studied for years was the large number of vessicles in endothelial cells seen in EM studies, the majority of which turned out to be derived from the fixation process. 

Carl

 

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Wednesday, March 26, 2008 3:42 PM

Subject: Re: Subnuclear localization of transcription factors in fixed tissue

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Confocalists,

Thanks a lot for your useful suggestions and the plenitude of great references. I am still digging through them.

I will be definitely testing whether my protein foci colocalize with the markers for the known subnuclear structures, such as  the "speckles" (or "interchromatin granule clusters" in EM), Cajal  bodies and PML bodies.

Some of you have raised a very valid point: that protein aggregates might be a result of an over-expression of a protein at abnormally high levels. This is not the case in our system: most of the times, we detect the endogenous protein in embryos. The antibodies we work with are specific, as they detect the Hox proteins in the regions where their genes are transcribed.

Thank you very much again,

Ella

-- 

Ella Tour
Department of Cell and Developmental Biology, 0349                    
University of California, San Diego            
9500 Gilman Drive, 4305 Bonner Hall                            
La Jolla, CA  92093-0349
Phone [phone]
FAX [phone]
email: [hidden email]

[Jeremy Adler]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

re suggestion of fixation artefacts, can you provide a useful reference ?

Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden



-----Original Message-----
From: Confocal Microscopy List on behalf of Carl Boswell
Sent: Thu 3/27/2008 01:11
To: [hidden email]
Subject: Re: Subnuclear localization of transcription factors in fixed tissue
 
Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Re: Subnuclear localization of transcription factors in fiHi Ella,
One thing I didn't see mentioned in this thread was the possibility of aggregates or precipitates forming during fixation/denaturation.  It's the first thing I think of when I see the term "speckles" to describe a staining pattern.  A classic artifact studied for years was the large number of vessicles in endothelial cells seen in EM studies, the majority of which turned out to be derived from the fixation process.  
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
  ----- Original Message -----
  From: Ella Tour
  To: [hidden email]
  Sent: Wednesday, March 26, 2008 3:42 PM
  Subject: Re: Subnuclear localization of transcription factors in fixed tissue


  Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal 
  Dear Confocalists,

  Thanks a lot for your useful suggestions and the plenitude of great references. I am still digging through them.


  I will be definitely testing whether my protein foci colocalize with the markers for the known subnuclear structures, such as  the "speckles" (or "interchromatin granule clusters" in EM), Cajal  bodies and PML bodies.

  Some of you have raised a very valid point: that protein aggregates might be a result of an over-expression of a protein at abnormally high levels. This is not the case in our system: most of the times, we detect the endogenous protein in embryos. The antibodies we work with are specific, as they detect the Hox proteins in the regions where their genes are transcribed.

  Thank you very much again,

  Ella


--




  Ella Tour
  Department of Cell and Developmental Biology, 0349                    
  University of California, San Diego            
  9500 Gilman Drive, 4305 Bonner Hall                            
  La Jolla, CA  92093-0349
  Phone [phone]
  FAX [phone]
  email: [hidden email]

[Boswell, Carl A - (cboswell)]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Sorry.  It was close to 20 years ago and I've moved on to other interests
and left that material behind.  It just remains in the back of my mind and
rises to the surface when the mention of tiny structures in fixed material
is discussed.  The vessicle artifact in endothelial cells might have been
identifed in the late 80's, if that helps.

Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
----- Original Message -----
From: "Jeremy Adler" <[hidden email]>
To: <[hidden email]>
Sent: Wednesday, March 26, 2008 10:31 PM
Subject: Re: Subnuclear localization of transcription factors in fixed
tissue


Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

re suggestion of fixation artefacts, can you provide a useful reference ?

Jeremy Adler
Cell Biology
The Wenner-Gren Inst.
Arrhenius Laboratories E5
Stockholm University
Stockholm 106 91
Sweden



-----Original Message-----
From: Confocal Microscopy List on behalf of Carl Boswell
Sent: Thu 3/27/2008 01:11
To: [hidden email]
Subject: Re: Subnuclear localization of transcription factors in fixed
tissue

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Re: Subnuclear localization of transcription factors in fiHi Ella,
One thing I didn't see mentioned in this thread was the possibility of
aggregates or precipitates forming during fixation/denaturation.  It's the
first thing I think of when I see the term "speckles" to describe a staining
pattern.  A classic artifact studied for years was the large number of
vessicles in endothelial cells seen in EM studies, the majority of which
turned out to be derived from the fixation process.
Carl

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]
  ----- Original Message -----
  From: Ella Tour
  To: [hidden email]
  Sent: Wednesday, March 26, 2008 3:42 PM
  Subject: Re: Subnuclear localization of transcription factors in fixed
tissue


  Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
  Dear Confocalists,

  Thanks a lot for your useful suggestions and the plenitude of great
references. I am still digging through them.


  I will be definitely testing whether my protein foci colocalize with the
markers for the known subnuclear structures, such as  the "speckles" (or
"interchromatin granule clusters" in EM), Cajal  bodies and PML bodies.

  Some of you have raised a very valid point: that protein aggregates might
be a result of an over-expression of a protein at abnormally high levels.
This is not the case in our system: most of the times, we detect the
endogenous protein in embryos. The antibodies we work with are specific, as
they detect the Hox proteins in the regions where their genes are
transcribed.

  Thank you very much again,

  Ella


--




  Ella Tour
  Department of Cell and Developmental Biology, 0349
  University of California, San Diego
  9500 Gilman Drive, 4305 Bonner Hall
  La Jolla, CA  92093-0349
  Phone [phone]
  FAX [phone]
  email: [hidden email]

[Ella Tour]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Carl,

Thank you for mentioning the fixation/denaturation artifacts, I've been wondering about that as well. I am familiar with the paper by Melan and Sudler (also mentioned in Jason Swdlow's reply, http://www.ncbi.nlm.nih.gov/pubmed/1527176). This paper demonstrates that localization of injected FITC-labeled proteins changes as a result of different methods of cell permeabilization, with worst artifacts observed for protocols where permeabilization with TritonX was done before fixation, the higher  TritonX concentrations, the worse the redistribution. When cells were fixed first  (3.7% formaldehyde, 30 min) and then permeabilized (0.1% TritonX, 10 min), protein distribution became less even and more punctate.

Now, as far as I know, most immunofluorescent studies of nuclear proteins employ the later protocol (2-4% formaldehyde fixation, permeabilization with 0.1-0.3% TritonX). Many of these studies show good colocalization between proteins belonging to the same nuclear structures (localization of splicing factors to "speckles", for example). So, at least for some proteins, the subnuclear localization seems to be decently preserved under these conditions. Is there a consensus as to whether this common fixation/permeabilization method causes significant changes in protein localization within the nucleus?

With regards to denaturation: often, we combine protein stains with DNA in situ hybridization. The DNA FISH protocol calls for heating of the tissue, in order to denaturate the embryonic DNA before the addition of the DNA probe. So, before detecting the protein, I subject the embryos to 15 min of 85 degrees Celsius and then an overnight hybridization in 50% formamide at 37 degrees Celsius. After that, we wash the probe with solutions containing 0.3% CHAPS ( a zwitterionic detergent). Any red flags here?

 

Thank you,

Ella

Hi Ella,

One thing I didn't see mentioned in this thread was the possibility of aggregates or precipitates forming during fixation/denaturation.  It's the first thing I think of when I see the term "speckles" to describe a staining pattern.  A classic artifact studied for years was the large number of vessicles in endothelial cells seen in EM studies, the majority of which turned out to be derived from the fixation process. 

Carl

 

Carl A. Boswell, Ph.D.
Molecular and Cellular Biology
University of Arizona
520-954-7053
FAX [phone]

----- Original Message -----

From: [hidden email]

To: [hidden email]

Sent: Wednesday, March 26, 2008 3:42 PM

Subject: Re: Subnuclear localization of transcription factors in fixed tissue

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal BLOCKQUOTE {     PADDING-BOTTOM: 0px; PADDING-TOP: 0px } DL {    PADDING-BOTTOM: 0px; PADDING-TOP: 0px } UL {    PADDING-BOTTOM: 0px; PADDING-TOP: 0px } OL {    PADDING-BOTTOM: 0px; PADDING-TOP: 0px } LI {    PADDING-BOTTOM: 0px; PADDING-TOP: 0px }

Dear Confocalists,

Thanks a lot for your useful suggestions and the plenitude of great references. I am still digging through them.

I will be definitely testing whether my protein foci colocalize with the markers for the known subnuclear structures, such as  the "speckles" (or "interchromatin granule clusters" in EM), Cajal  bodies and PML bodies.

Some of you have raised a very valid point: that protein aggregates might be a result of an over-expression of a protein at abnormally high levels. This is not the case in our system: most of the times, we detect the endogenous protein in embryos. The antibodies we work with are specific, as they detect the Hox proteins in the regions where their genes are transcribed.

Thank you very much again,

Ella

--

Ella Tour
Department of Cell and Developmental Biology, 0349                    
University of California, San Diego            

9500 Gilman Drive, 4305 Bonner Hall                            
La Jolla, CA  92093-0349
Phone [phone]
FAX [phone]
email: [hidden email]

-- 

Ella Tour
Department of Cell and Developmental Biology, 0349                    
University of California, San Diego            
9500 Gilman Drive, 4305 Bonner Hall                            
La Jolla, CA  92093-0349
Phone [phone]
FAX [phone]
email: [hidden email]

[Patrick Van Oostveldt]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi,

I think the combination of FISH and protein immuno localisation is a  
ever lasting problem which needs multiple checks.
A question in this is the presence of RNA as a linking and structuring  
element in the cell nucleus. In order to reach high specificity for  
FISH it is adviced to do a RNAase treatment, and even some protease  
treatment.
Further more, does a treatment with formamide not alter your epitope  
and affects specificity of the immuno-detection?
A fundamental question here is: how can we include the necessary  
controls? Positive and negatives?
I really follow this discussion with high interest.

Patrick Van Oostveldt

Quoting Ella Tour <[hidden email]>:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Hi Carl,
>
> Thank you for mentioning the fixation/denaturation artifacts, I've been
> wondering about that as well. I am familiar with the paper by Melan and
> Sudler (also mentioned in Jason Swdlow's reply,
> http://www.ncbi.nlm.nih.gov/pubmed/1527176). This paper demonstrates
> that localization of injected FITC-labeled proteins changes as a result
> of different methods of cell permeabilization, with worst artifacts
> observed for protocols where permeabilization with TritonX was done
> before fixation, the higher  TritonX concentrations, the worse the
> redistribution. When cells were fixed first  (3.7% formaldehyde, 30
> min) and then permeabilized (0.1% TritonX, 10 min), protein
> distribution became less even and more punctate.
>
> Now, as far as I know, most immunofluorescent studies of nuclear
> proteins employ the later protocol (2-4% formaldehyde fixation,
> permeabilization with 0.1-0.3% TritonX). Many of these studies show
> good colocalization between proteins belonging to the same nuclear
> structures (localization of splicing factors to "speckles", for
> example). So, at least for some proteins, the subnuclear localization
> seems to be decently preserved under these conditions. Is there a
> consensus as to whether this common fixation/permeabilization method
> causes significant changes in protein localization within the nucleus?
>
> With regards to denaturation: often, we combine protein stains with DNA
> in situ hybridization. The DNA FISH protocol calls for heating of the
> tissue, in order to denaturate the embryonic DNA before the addition of
> the DNA probe. So, before detecting the protein, I subject the embryos
> to 15 min of 85 degrees Celsius and then an overnight hybridization in
> 50% formamide at 37 degrees Celsius. After that, we wash the probe with
> solutions containing 0.3% CHAPS ( a zwitterionic detergent). Any red
> flags here?
>
> Thank you,
>
> Ella
>
>
>>
>> Hi Ella,
>> One thing I didn't see mentioned in this thread was the possibility  
>>  of aggregates or precipitates forming during  
>> fixation/denaturation.  It's the first thing I think of when I see  
>> the term "speckles" to  describe a staining pattern.  A classic  
>> artifact studied for years  was the large number of vessicles in  
>> endothelial cells seen in EM  studies, the majority of which turned  
>> out to be derived from the  fixation process. Carl
>>
>> Carl A. Boswell, Ph.D.
>> Molecular and Cellular Biology
>> University of Arizona
>> 520-954-7053
>> FAX [phone]
>>
>> ----- Original Message -----
>> From: <mailto:[hidden email]>Ella Tour
>> To: <mailto:[hidden email]>[hidden email]
>> Sent: Wednesday, March 26, 2008 3:42 PM
>> Subject: Re: Subnuclear localization of transcription factors in  
>> fixed tissue
>>
>> Search the CONFOCAL archive at  
>> <http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal BLOCKQUOTE { PADDING-BOTTOM: 0px; PADDING-TOP: 0px } DL { PADDING-BOTTOM: 0px; PADDING-TOP: 0px } UL { PADDING-BOTTOM: 0px; PADDING-TOP: 0px } OL { PADDING-BOTTOM: 0px; PADDING-TOP: 0px } LI { PADDING-BOTTOM: 0px; PADDING-TOP: 0px  
>> }
>> Dear Confocalists,
>>
>> Thanks a lot for your useful suggestions and the plenitude of great  
>>  references. I am still digging through them.
>>
>> I will be definitely testing whether my protein foci colocalize  
>> with the markers for the known subnuclear structures, such as  the  
>> "speckles" (or "interchromatin granule clusters" in EM), Cajal  
>> bodies and PML bodies.
>>
>> Some of you have raised a very valid point: that protein aggregates  
>>  might be a result of an over-expression of a protein at abnormally  
>>  high levels. This is not the case in our system: most of the  
>> times,  we detect the endogenous protein in embryos. The antibodies  
>> we work  with are specific, as they detect the Hox proteins in the  
>> regions  where their genes are transcribed.
>>
>> Thank you very much again,
>>
>> Ella
>>
>> --
>>
>>
>>
>>
>> Ella Tour
>> Department of Cell and Developmental Biology, 0349
>> University of California, San Diego
>> 9500 Gilman Drive, 4305 Bonner Hall
>> La Jolla, CA  92093-0349
>> Phone [phone]
>> FAX [phone]
>> email: [hidden email]
>
>
> --
>
>
>
>
> Ella Tour
> Department of Cell and Developmental Biology, 0349
> University of California, San Diego
> 9500 Gilman Drive, 4305 Bonner Hall
> La Jolla, CA  92093-0349
> Phone [phone]
> FAX [phone]
> email: [hidden email]

--
Dep. Moleculaire Biotechnologie
Coupure links 653
B 9000 GENT

tel [phone]
fax [phone]

[Monique Vasseur]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear all,

We are using an Eppendorf FemtoJet to inject femtoliters of solution in living cells.  The instruction manual says you multiply the pressure by the injection time to get the volume injected...  Pressure is in hPa and time in second.  I don't have any idea to what hPa corresponds and what should I get as a result: microliters? femtoliters?  Is there other variable to put in the equation to get the volume (we use FemtoTips)?

Thanks a lot for your help.
 
Monique Vasseur
Microscopie et imagerie
Département de biochimie
Université de Montréal
C.P. 6128, succursale Centre-ville
Montréal QC    H3C 3J7   Canada
tél. [phone] poste 5148

[Julio Vazquez]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal =

Hello Monique, 

The pressure determines the flow (volume of liquid passing through the pipet per unit of time). This Volume depends on the pressure differential (pressure applied to liquid - pressure inside cell), the viscosity of the liquid, and the diameter and length of the pipet. The actual volume injected will be equal to the flow rate multiplied by the time. I think the Hagen-Poiseuille law is the one that describes this:

delta P = 8 u L Q / Pi r^4, where P is pressure, u is viscosity, Q is flow rate, L is the length of the tube, and r is the radius.  From this you can see that the flow rate is proportional to the pressure:  Q = delta P Pi r^4 / 8 u L . For any given set-up, Pi r^4/8 u L is a constant (K) so Flow = (Pressure) x (K), and Volume injected = (Flow) x (Time). To get the actual volume, you need to know all the parameters (diameter, length, viscosity, pressure differential). However, if you consider that all these are fixed parameters, then the volume injected varies as a function of the only two variables pressure applied and injection time. 

For a more accurate and detailed description, you can check Wikipedia:

http://en.wikipedia.org/wiki/Hagen-Poiseuille_law

--

Julio Vazquez, PhD

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,  mailstop DE-512

Seattle, WA 98109-1024

http://www.fhcrc.org

==

On Mar 28, 2008, at 7:02 AM, Vasseur Monique wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear all,

We are using an Eppendorf FemtoJet to inject femtoliters of solution in living cells.  The instruction manual says you multiply the pressure by the injection time to get the volume injected...  Pressure is in hPa and time in second.  I don't have any idea to what hPa corresponds and what should I get as a result: microliters? femtoliters?  Is there other variable to put in the equation to get the volume (we use FemtoTips)?

Thanks a lot for your help.

Monique Vasseur

Microscopie et imagerie

Département de biochimie

Université de Montréal

C.P. 6128, succursale Centre-ville

Montréal QC    H3C 3J7   Canada

tél. [phone] poste 5148

[Ella Tour]

Search the CONFOCAL archive at
http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi Patrick,

I agree that multiple tests need to be done in order to establish
that protein localization is preserved during the various treatments.
Your point about the need for RNA as a structural element: since I am
working with relatively small probes against enhancers, which seem
not to have a lot of transcription units in them, I've been doing DNA
FISH/protein stain combo with or without the RNAse treatment. I don't
see dramatic changes in protein distribution with or without the
RNAse treatment. However, this is on a very gross level, I agree that
it is possible that some foci might change their location relatively
to other nuclear structures or genes.
Same for the treatment with formamide: I don't see gross changes in
protein distribution in my DNA/protein combo versus just protein
immunodetection, but then again, the changes might be subtle.

One control I am trying to get to work right now is taking a
well-identified transcription factor - regulatory sequence system,
where the localization of the TF to its target sequence has been
shown in vivo, and seeing that this localization can be detected via
IF in my system. An example of such is Lac Repressor (LacI)-GFP
fusion and its target sequence, multiple copies of Lac Operon sites
(LacO). The visualization of LacI-GFP on LacO repeats has been
described in a number of papers (for example, Vazquez et al (2006)
Mol Bio Cell, where the clustering of LacI-GFP molecules on 64 LacO
sites was very nicely shown).  I assume that, since Alexa fluors are
brighter than GFP, I should be able to detect clustering of LacI-GFP
on at least 64 LacO sites? I'd appreciate some feedback on this one
from people who have experience with GFP detection in vivo and via IF
.

Thank you,

Ella

>Hi,
>
>I think the combination of FISH and protein immuno localisation is a
>ever lasting problem which needs multiple checks.
>A question in this is the presence of RNA as a linking and
>structuring element in the cell nucleus. In order to reach high
>specificity for FISH it is adviced to do a RNAase treatment, and
>even some protease treatment.
>Further more, does a treatment with formamide not alter your epitope
>and affects specificity of the immuno-detection?
>A fundamental question here is: how can we include the necessary
>controls? Positive and negatives?
>I really follow this discussion with high interest.
>
>Patrick Van Oostveldt
>
>Quoting Ella Tour <[hidden email]>:
>
>>Search the CONFOCAL archive at
>>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>>
>>Hi Carl,
>>
>>Thank you for mentioning the fixation/denaturation artifacts, I've been
>>wondering about that as well. I am familiar with the paper by Melan and
>>Sudler (also mentioned in Jason Swdlow's reply,
>>http://www.ncbi.nlm.nih.gov/pubmed/1527176). This paper demonstrates
>>that localization of injected FITC-labeled proteins changes as a result
>>of different methods of cell permeabilization, with worst artifacts
>>observed for protocols where permeabilization with TritonX was done
>>before fixation, the higher  TritonX concentrations, the worse the
>>redistribution. When cells were fixed first  (3.7% formaldehyde, 30
>>min) and then permeabilized (0.1% TritonX, 10 min), protein
>>distribution became less even and more punctate.
>>
>>Now, as far as I know, most immunofluorescent studies of nuclear
>>proteins employ the later protocol (2-4% formaldehyde fixation,
>>permeabilization with 0.1-0.3% TritonX). Many of these studies show
>>good colocalization between proteins belonging to the same nuclear
>>structures (localization of splicing factors to "speckles", for
>>example). So, at least for some proteins, the subnuclear localization
>>seems to be decently preserved under these conditions. Is there a
>>consensus as to whether this common fixation/permeabilization method
>>causes significant changes in protein localization within the nucleus?
>>
>>With regards to denaturation: often, we combine protein stains with DNA
>>in situ hybridization. The DNA FISH protocol calls for heating of the
>>tissue, in order to denaturate the embryonic DNA before the addition of
>>the DNA probe. So, before detecting the protein, I subject the embryos
>>to 15 min of 85 degrees Celsius and then an overnight hybridization in
>>50% formamide at 37 degrees Celsius. After that, we wash the probe with
>>solutions containing 0.3% CHAPS ( a zwitterionic detergent). Any red
>>flags here?
>>
>>Thank you,
>>
>>Ella
>>
>>>
>>>Hi Ella,
>>>One thing I didn't see mentioned in this thread was the
>>>possibility  of aggregates or precipitates forming during
>>>fixation/denaturation.  It's the first thing I think of when I see
>>>the term "speckles" to  describe a staining pattern.  A classic
>>>artifact studied for years  was the large number of vessicles in
>>>endothelial cells seen in EM  studies, the majority of which
>>>turned out to be derived from the  fixation process. Carl
>>>
>>>Carl A. Boswell, Ph.D.
>>>Molecular and Cellular Biology
>>>University of Arizona
>>>520-954-7053
>>>FAX [phone]

--




Ella Tour
Department of Cell and Developmental Biology, 0349
University of California, San Diego
9500 Gilman Drive, 4305 Bonner Hall
La Jolla, CA  92093-0349
Phone [phone]
FAX [phone]
email: [hidden email]

[Monique Vasseur]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello Julio

 

Thanks a lot for the clarification.  By the same way, is there a database where internal cell pressures are listed?  Actually we are more interested in cardiomyocyte cytoplasm.

 

Monique Vasseur

Microscopie et imagerie

Département de biochimie

Université de Montréal

C.P. 6128, succursale Centre-ville

Montréal QC    H3C 3J7   Canada

tél. [phone] poste 5148

---

De : Confocal Microscopy List [mailto:[hidden email]] De la part de Julio Vazquez
Envoyé : 28 mars 2008 12:52
À : [hidden email]
Objet : Re: hPa injection pressure and volume calculation

 

=

Hello Monique, 

 

The pressure determines the flow (volume of liquid passing through the pipet per unit of time). This Volume depends on the pressure differential (pressure applied to liquid - pressure inside cell), the viscosity of the liquid, and the diameter and length of the pipet. The actual volume injected will be equal to the flow rate multiplied by the time. I think the Hagen-Poiseuille law is the one that describes this:

 

 

delta P = 8 u L Q / Pi r^4, where P is pressure, u is viscosity, Q is flow rate, L is the length of the tube, and r is the radius.  From this you can see that the flow rate is proportional to the pressure:  Q = delta P Pi r^4 / 8 u L . For any given set-up, Pi r^4/8 u L is a constant (K) so Flow = (Pressure) x (K), and Volume injected = (Flow) x (Time). To get the actual volume, you need to know all the parameters (diameter, length, viscosity, pressure differential). However, if you consider that all these are fixed parameters, then the volume injected varies as a function of the only two variables pressure applied and injection time. 

 

 

For a more accurate and detailed description, you can check Wikipedia:

 

http://en.wikipedia.org/wiki/Hagen-Poiseuille_law

 

 

 

 

 

--

Julio Vazquez, PhD

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,  mailstop DE-512

Seattle, WA 98109-1024

 

 

http://www.fhcrc.org

 

==

 

On Mar 28, 2008, at 7:02 AM, Vasseur Monique wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

 

Dear all,

 

We are using an Eppendorf FemtoJet to inject femtoliters of solution in living cells.  The instruction manual says you multiply the pressure by the injection time to get the volume injected...  Pressure is in hPa and time in second.  I don't have any idea to what hPa corresponds and what should I get as a result: microliters? femtoliters?  Is there other variable to put in the equation to get the volume (we use FemtoTips)?

 

Thanks a lot for your help.

 

Monique Vasseur

Microscopie et imagerie

Département de biochimie

Université de Montréal

C.P. 6128, succursale Centre-ville

Montréal QC    H3C 3J7   Canada

tél. [phone] poste 5148

 

[Julio Vazquez]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal =

Monique, 

I do not know if there is a database with such info, and don't have a number to give you. However, when I used to microinject Drosophila embryos, there was a minimal pressure that one would need to use in order for the solution to get inside the embryo (as opposed to fluid from the embryo feeding back into the pipet). I am guessing you could probably do a test where you take a sample solution identical to the one you would normally inject, add a tracer dye, and then see what minimal pressure you would need so that your colored solution gets inside the cell. This would give you a baseline pressure (basically, you have equilibrium between micropipet pressure and cell pressure when there is no net flow of your solution.  Your delta P then would be the difference between whatever pressure setting you are using for microinjection and this baseline value.  This is easy to do with embryos, because the volumes are relatively large and one can see liquid flowing...not sure if this is applicable to cells, but should be pretty easy to try if you already have the injection set-up working. 

Julio.

On Mar 28, 2008, at 11:37 AM, Vasseur Monique wrote:

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hello Julio

 

Thanks a lot for the clarification.  By the same way, is there a database where internal cell pressures are listed?  Actually we are more interested in cardiomyocyte cytoplasm.

 

Monique Vasseur

Microscopie et imagerie

Département de biochimie

Université de Montréal

C.P. 6128, succursale Centre-ville

Montréal QC    H3C 3J7   Canada

tél. [phone] poste 5148

---

De : Confocal Microscopy List [[hidden email]] De la part de Julio Vazquez
Envoyé : 28 mars 2008 12:52
À : [hidden email]
Objet : Re: hPa injection pressure and volume calculation

 

=

Hello Monique, 

 

The pressure determines the flow (volume of liquid passing through the pipet per unit of time). This Volume depends on the pressure differential (pressure applied to liquid - pressure inside cell), the viscosity of the liquid, and the diameter and length of the pipet. The actual volume injected will be equal to the flow rate multiplied by the time. I think the Hagen-Poiseuille law is the one that describes this:

 

 

delta P = 8 u L Q / Pi r^4, where P is pressure, u is viscosity, Q is flow rate, L is the length of the tube, and r is the radius.  From this you can see that the flow rate is proportional to the pressure:  Q = delta P Pi r^4 / 8 u L . For any given set-up, Pi r^4/8 u L is a constant (K) so Flow = (Pressure) x (K), and Volume injected = (Flow) x (Time). To get the actual volume, you need to know all the parameters (diameter, length, viscosity, pressure differential). However, if you consider that all these are fixed parameters, then the volume injected varies as a function of the only two variables pressure applied and injection time. 

 

 

For a more accurate and detailed description, you can check Wikipedia:

 

http://en.wikipedia.org/wiki/Hagen-Poiseuille_law

 

 

 

 

 

--

Julio Vazquez, PhD

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,  mailstop DE-512

Seattle, WA 98109-1024

 

 

http://www.fhcrc.org

 

==

 

On Mar 28, 2008, at 7:02 AM, Vasseur Monique wrote:

Search the CONFOCAL archive at

http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

 

Dear all,

 

We are using an Eppendorf FemtoJet to inject femtoliters of solution in living cells.  The instruction manual says you multiply the pressure by the injection time to get the volume injected...  Pressure is in hPa and time in second.  I don't have any idea to what hPa corresponds and what should I get as a result: microliters? femtoliters?  Is there other variable to put in the equation to get the volume (we use FemtoTips)?

 

Thanks a lot for your help.

 

Monique Vasseur

Microscopie et imagerie

Département de biochimie

Université de Montréal

C.P. 6128, succursale Centre-ville

Montréal QC    H3C 3J7   Canada

tél. [phone] poste 5148

 

[Rietdorf, Jens]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Dear Monique,

 

from many years of experience I can tell you its very difficult to estimate the injected volume, which will also depend on the geometry of the pipet you use for injection. Very often the openings of these pipets are partially closed by dirt, cell debris or aggregates in the injected fluid.   The best way to get a rough idea is to co-inject a fluorescently labeled dextran. Variations are huge, if you compare cells of similar size injected with the same pipet, pressure etc you will see these appear very different in brightness after injection. Injecting into low viscosity mineral oil can help to estimate the volume by measuring the size of the droplets in the oil.

 

Good luck, jens 

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From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Julio Vazquez
Sent: Freitag, 28. März 2008 17:52
To: [hidden email]
Subject: [SPAM] Re: hPa injection pressure and volume calculation

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal =

Hello Monique, 

The pressure determines the flow (volume of liquid passing through the pipet per unit of time). This Volume depends on the pressure differential (pressure applied to liquid - pressure inside cell), the viscosity of the liquid, and the diameter and length of the pipet. The actual volume injected will be equal to the flow rate multiplied by the time. I think the Hagen-Poiseuille law is the one that describes this:

delta P = 8 u L Q / Pi r^4, where P is pressure, u is viscosity, Q is flow rate, L is the length of the tube, and r is the radius.  From this you can see that the flow rate is proportional to the pressure:  Q = delta P Pi r^4 / 8 u L . For any given set-up, Pi r^4/8 u L is a constant (K) so Flow = (Pressure) x (K), and Volume injected = (Flow) x (Time). To get the actual volume, you need to know all the parameters (diameter, length, viscosity, pressure differential). However, if you consider that all these are fixed parameters, then the volume injected varies as a function of the only two variables pressure applied and injection time. 

For a more accurate and detailed description, you can check Wikipedia:

http://en.wikipedia.org/wiki/Hagen-Poiseuille_law

--

Julio Vazquez, PhD

Fred Hutchinson Cancer Research Center

1100 Fairview Ave. N.,  mailstop DE-512

Seattle, WA 98109-1024

http://www.fhcrc.org

==

On Mar 28, 2008, at 7:02 AM, Vasseur Monique wrote:

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Dear all,

We are using an Eppendorf FemtoJet to inject femtoliters of solution in living cells.  The instruction manual says you multiply the pressure by the injection time to get the volume injected...  Pressure is in hPa and time in second.  I don't have any idea to what hPa corresponds and what should I get as a result: microliters? femtoliters?  Is there other variable to put in the equation to get the volume (we use FemtoTips)?

Thanks a lot for your help.

Monique Vasseur

Microscopie et imagerie

Département de biochimie

Université de Montréal

C.P. 6128, succursale Centre-ville

Montréal QC    H3C 3J7   Canada

tél. [phone] poste 5148