Acceptor Photobleaching vs. Sensitized Emission FRET results

[Pablo German]

Dear list members,

I have been doing some FRET microscopy experiments on a
7-TransMembrane domain receptor tagged with either eCFP/eYFP at the
different intra-cellular loops (ICL1, ICL2, and ICL3). I have tried
the 9 different combinations (e.g ICL1-YFP + ICL1-CFP, ICL1-YFP +
ICL2-CFP, etc) to see if I could detect any difference in FRET
efficiency.

I have anlyzed the images by both Sensitized Emission and Acceptor
Photobleaching using the pFRET plugin on ImageJ developed at KCCI-UVa.
The problem is the following: the results using Sensitized Emission
give me significant differences between the different pairs but the
results using APB give me no differences (all about 25% efficiency).

I have the feeling that I should trust APB more than SE. I have
noticed that, when using SE, the higher the difference in intensity
between YFP and CFP, the higher the FRET efficiency.

Has anyone had a similar experience? Which method of analysis should I trust?

Regards,
Pablo

--
Pablo German
PhD Candidate

Plant and Food Research
Private Bag 92169
Auckland Mail Centre
Auckland 1142
New Zealand
DDI: [phone]
Mobile: [phone]

[yuansheng sun]

Dear Pablo,

This is sheng, working at the Keck center, UVA. I think we have
contacted earlier for the new pFRET software. Here are my comments for
this topic:

I would not be surprised to see different FRET efficiencies for
different Donor : Acceptor ratios or different Acceptor levels. That
is actually a valuable indication for the random association or the
cluster assembly. I recommend you look at the following paper -
Biophys. J. Vol 85, Issue 1, 559-571 (2003).
If you write me an email ([hidden email]), I can send you a
couple of nice PPTs on this topic.

I like your strategy - measuring FRET of a same system in different
ways (SE vs. AP). If possible, I would like to try FLIM as well since
lifetime is independent of fluorophore concentration, only if
possible. We have to work with what we have. I would not make the
decision to accept AP and reject SE, because I do not see a reason why
AP can give you more accurate (quantitative) results than SE, if your
experiments were done properly.

There are actually some potential issues you may check for using AP. I
assume your measurements were done with live cells.

1. Check if the donor is also bleached during the photobleaching
process. Use the donor-alone specimen to check. The apFRET plugin in
the new pFRET software allows you correct for this issue.

2. Check if the acceptor is completely bleached. Take the pre- and
post- acceptor images. Your FRET efficiency is certainly influenced by
the left acceptor amount after photobleaching. The apFRET plugin in
the new pFRET software allows you address this issue.

3. Check if there is any cellular movement or focus change. Overlay
pre- and post- images in two different colors to see if you will have
a perfect overlay. If not, I suggest you run AP with fixed cells to
see you will also have homogeneous FRET efficiencies.

Please shoot me an email if you need help using the pFRET software to
check the issues mentioned above. Good luck.

Best regards,
sheng




On Tue, Mar 30, 2010 at 11:53 PM, Pablo German
<[hidden email]> wrote:

> Dear list members,
>
> I have been doing some FRET microscopy experiments on a
> 7-TransMembrane domain receptor tagged with either eCFP/eYFP at the
> different intra-cellular loops (ICL1, ICL2, and ICL3). I have tried
> the 9 different combinations (e.g ICL1-YFP + ICL1-CFP, ICL1-YFP +
> ICL2-CFP, etc) to see if I could detect any difference in FRET
> efficiency.
>
> I have anlyzed the images by both Sensitized Emission and Acceptor
> Photobleaching using the pFRET plugin on ImageJ developed at KCCI-UVa.
> The problem is the following: the results using Sensitized Emission
> give me significant differences between the different pairs but the
> results using APB give me no differences (all about 25% efficiency).
>
> I have the feeling that I should trust APB more than SE. I have
> noticed that, when using SE, the higher the difference in intensity
> between YFP and CFP, the higher the FRET efficiency.
>
> Has anyone had a similar experience? Which method of analysis should I trust?
>
> Regards,
> Pablo
>
> --
> Pablo German
> PhD Candidate
>
> Plant and Food Research
> Private Bag 92169
> Auckland Mail Centre
> Auckland 1142
> New Zealand
> DDI: [phone]
> Mobile: [phone]
>

[yuansheng sun]

Pablo,

I forgot to ask if the FRET efficiency you mentioned refers to the
average of the whole cell. I think it is more appreciate to do
quantitative comparisons in details, such as comparing the FRET
efficiencies of different pairs for the same Donor : Acceptor ratios
or the same acceptor levels. If you can write me more details, we can
further discuss about the data analysis strategy.

sheng


On Tue, Mar 30, 2010 at 11:53 PM, Pablo German
<[hidden email]> wrote:

> Dear list members,
>
> I have been doing some FRET microscopy experiments on a
> 7-TransMembrane domain receptor tagged with either eCFP/eYFP at the
> different intra-cellular loops (ICL1, ICL2, and ICL3). I have tried
> the 9 different combinations (e.g ICL1-YFP + ICL1-CFP, ICL1-YFP +
> ICL2-CFP, etc) to see if I could detect any difference in FRET
> efficiency.
>
> I have anlyzed the images by both Sensitized Emission and Acceptor
> Photobleaching using the pFRET plugin on ImageJ developed at KCCI-UVa.
> The problem is the following: the results using Sensitized Emission
> give me significant differences between the different pairs but the
> results using APB give me no differences (all about 25% efficiency).
>
> I have the feeling that I should trust APB more than SE. I have
> noticed that, when using SE, the higher the difference in intensity
> between YFP and CFP, the higher the FRET efficiency.
>
> Has anyone had a similar experience? Which method of analysis should I trust?
>
> Regards,
> Pablo
>
> --
> Pablo German
> PhD Candidate
>
> Plant and Food Research
> Private Bag 92169
> Auckland Mail Centre
> Auckland 1142
> New Zealand
> DDI: [phone]
> Mobile: [phone]
>

[Louis Villeneuve]

Bonjour à tous,

We are trying to (indirect) immunostain with antiboby on  heart cryosections (14um).  The proteins that we want to identified are from the cytoskeleton like desmin, myosin light chain, myosin heavy chain.  We have a problem that all the staining stay on top of the tissue for those antibodies.  Counterstaining for actin (phalloidin alexa conjugated) is pretty good through the thickness of the tissue.  We permealized the tissue with Triton 0.5% , in the blocking solution (serum from the host of the 2nd Ab), for 1 hour and we incubate the primary overnight at  4C (with Triton 0.2% in the antibody diluent).  Other antibodies against cytoskeleton protein (myosin light chain 7) are well stained over the thickness of the tissue using the same protocol and the same batch of tissues.

Any clue that might help us?

Louissssssss
Louis Villeneuve
Research Associate- Confocal Microscopy
Heart Montreal Institute- Research Center
5000 East Belanger
Montreal (Qc), Canada
H1T 1C8

514-376-3330 ext 3511
514-376-1355 (Fax)

[hidden email]

[Martin Wessendorf-2]

Dear Louis--

It might simply be that the antibodies don't penetrate well--some don't.
  In my experience, cryostat sections are more problematic than sections
that are stained free-floating and mounted on slides after the staining.
  However, some antibodies appear just not to diffuse through tissue
easily.

Good luck!

Martin Wessendorf

[hidden email] wrote:

>
> Bonjour à tous,
>
> We are trying to (indirect) immunostain with antiboby on  heart
> cryosections (14um).  The proteins that we want to identified are from
> the cytoskeleton like desmin, myosin light chain, myosin heavy chain.
>  We have a problem that all the staining stay on top of the tissue for
> those antibodies.  Counterstaining for actin (phalloidin alexa
> conjugated) is pretty good through the thickness of the tissue.  We
> permealized the tissue with Triton 0.5% , in the blocking solution
> (serum from the host of the 2nd Ab), for 1 hour and we incubate the
> primary overnight at  4C (with Triton 0.2% in the antibody diluent).
>  Other antibodies against cytoskeleton protein (myosin light chain 7)
> are well stained over the thickness of the tissue using the same
> protocol and the same batch of tissues.
>
> Any clue that might help us?
>
> Louissssssss
> Louis Villeneuve
> Research Associate- Confocal Microscopy
> Heart Montreal Institute- Research Center
> 5000 East Belanger
> Montreal (Qc), Canada
> H1T 1C8
>
> 514-376-3330 ext 3511
> 514-376-1355 (Fax)
>
> [hidden email]

--
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]

[RICHARD BURRY]

Louis

 

One problem could be rinses after antibody incubations.  I have recently did experiments published in my Springer book” Immunocytochemistry – A Practical Guide for Biomedical Research”, looking at labeling after different numbers of rinses.  A low number of rinses after a primary antibody incubation greatly reduced labeling with no background.  The primary antibody on the sections was reacting with the secondary antibody in solution and preventing its penetration into the tissue.  The effect was that the concentration of the unbound secondary antibody was much lower than that added.  I found that 7 rinses were needed for the conditions I used to gain maximum labeling with the secondary antibody.  However, it is the lack of rinses after secondary antibody that increases background.

 

Richard Burry

----- Original Message -----
From: [hidden email]
Date: Tuesday, April 20, 2010 4:50 pm
Subject: Lack of penetration of immunolabelling
To: [hidden email]


> Bonjour à tous,

> We are trying to (indirect) immunostain with antiboby on  heart cryosections (14um).  The proteins that we want to identified are from the cytoskeleton like desmin, myosin light chain, myosin heavy chain.  We have a problem that all the staining stay on top of the tissue for those antibodies.  Counterstaining for actin (phalloidin alexa conjugated) is pretty good through the thickness of the tissue.  We permealized the tissue with Triton 0.5% , in the blocking solution (serum from the host of the 2nd Ab), for 1 hour and we incubate the primary overnight at  4C (with Triton 0.2% in the antibody diluent).  Other antibodies against cytoskeleton protein (myosin light chain 7) are well stained over the thickness of the tissue using the same protocol and the same batch of tissues.

> Any clue that might help us?

> Louissssssss
> Louis Villeneuve
> Research Associate- Confocal Microscopy
> Heart Montreal Institute- Research Center
> 5000 East Belanger
> Montreal (Qc), Canada
> H1T 1C8

> 514-376-3330 ext 3511
> 514-376-1355 (Fax)

> [hidden email]

---

> Spam
> Not spam
> Forget previous vote



Richard W. Burry, Ph.D.
Department of Neuroscience, College of Medicine
Campus Microscopy and Imaging Facility, Director
The Ohio State University
Associate Editor, Journal of Histochemistry and Cytochemistry
277 Biomedical Research Tower
460 West Twelfth Avenue
Columbus, Ohio 43210
Voice 614.292.2814  Cell 614.638.3345  Fax [phone]

[Glen MacDonald-2]

What is the length of incubation for the secondary antibody?  The typical 1-2 hr. incubation tends to be too short for most tissue sections.  


Regards,
Glen

On Apr 20, 2010, at 1:45 PM, [hidden email] wrote:

>
> Bonjour à tous,
>
> We are trying to (indirect) immunostain with antiboby on  heart cryosections (14um).  The proteins that we want to identified are from the cytoskeleton like desmin, myosin light chain, myosin heavy chain.  We have a problem that all the staining stay on top of the tissue for those antibodies.  Counterstaining for actin (phalloidin alexa conjugated) is pretty good through the thickness of the tissue.  We permealized the tissue with Triton 0.5% , in the blocking solution (serum from the host of the 2nd Ab), for 1 hour and we incubate the primary overnight at  4C (with Triton 0.2% in the antibody diluent).  Other antibodies against cytoskeleton protein (myosin light chain 7) are well stained over the thickness of the tissue using the same protocol and the same batch of tissues.
>
> Any clue that might help us?
>
> Louissssssss
> Louis Villeneuve
> Research Associate- Confocal Microscopy
> Heart Montreal Institute- Research Center
> 5000 East Belanger
> Montreal (Qc), Canada
> H1T 1C8
>
> 514-376-3330 ext 3511
> 514-376-1355 (Fax)
>
> [hidden email]

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]

[Phillips, Thomas E.]

Another consideration is whether your primary is an IgM or IgG. IgM's are considerably bigger but a non-insignificant proportion of non-commercial primaries and even some commercial ones are IgM. Phalloidin's MW = 790 while an IgG is closer to 150,000 and an IgM 5x that.


Thomas E. Phillips, Ph.D
Professor of Biological Sciences
Director, Molecular Cytology Core
2 Tucker Hall
University of Missouri
Columbia, MO 65211-7400
573-882-4712 (office)
573-882-0123 (fax)
[hidden email]

http://www.biology.missouri.edu/faculty/phillips.html
http://www.biotech.missouri.edu/mcc/


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Glen MacDonald
Sent: Tuesday, April 20, 2010 5:07 PM
To: [hidden email]
Subject: Re: Lack of penetration of immunolabelling

What is the length of incubation for the secondary antibody?  The typical 1-2 hr. incubation tends to be too short for most tissue sections.  


Regards,
Glen

On Apr 20, 2010, at 1:45 PM, [hidden email] wrote:

>
> Bonjour à tous,
>
> We are trying to (indirect) immunostain with antiboby on  heart cryosections (14um).  The proteins that we want to identified are from the cytoskeleton like desmin, myosin light chain, myosin heavy chain.  We have a problem that all the staining stay on top of the tissue for those antibodies.  Counterstaining for actin (phalloidin alexa conjugated) is pretty good through the thickness of the tissue.  We permealized the tissue with Triton 0.5% , in the blocking solution (serum from the host of the 2nd Ab), for 1 hour and we incubate the primary overnight at  4C (with Triton 0.2% in the antibody diluent).  Other antibodies against cytoskeleton protein (myosin light chain 7) are well stained over the thickness of the tissue using the same protocol and the same batch of tissues.
>
> Any clue that might help us?
>
> Louissssssss
> Louis Villeneuve
> Research Associate- Confocal Microscopy
> Heart Montreal Institute- Research Center
> 5000 East Belanger
> Montreal (Qc), Canada
> H1T 1C8
>
> 514-376-3330 ext 3511
> 514-376-1355 (Fax)
>
> [hidden email]

Glen MacDonald
Core for Communication Research
Virginia Merrill Bloedel Hearing Research Center
Box 357923
University of Washington
Seattle, WA 98195-7923  USA
[phone]
[hidden email]

[Tamara Howard]

How were the samples fixed? Do you fix the sample & then
freeze, or are these fresh-frozen and then the sections
fixed? Are you certain that your problem antibodies are
compatible with the fixation?

Tamara

On Tue, 20 Apr 2010 16:45:37 -0400
  [hidden email] wrote:

> Bonjour à tous,
>
> We are trying to (indirect) immunostain with antiboby on
> heart
> cryosections (14um).  The proteins that we want to
>identified are from the
> cytoskeleton like desmin, myosin light chain, myosin
>heavy chain.  We have
> a problem that all the staining stay on top of the
>tissue for those
> antibodies.  Counterstaining for actin (phalloidin alexa
>conjugated) is
> pretty good through the thickness of the tissue.  We
>permealized the
> tissue with Triton 0.5% , in the blocking solution
>(serum from the host of
> the 2nd Ab), for 1 hour and we incubate the primary
>overnight at  4C (with
> Triton 0.2% in the antibody diluent).  Other antibodies
>against
> cytoskeleton protein (myosin light chain 7) are well
>stained over the
> thickness of the tissue using the same protocol and the
>same batch of
> tissues.
>
> Any clue that might help us?
>
> Louissssssss
> Louis Villeneuve
> Research Associate- Confocal Microscopy
> Heart Montreal Institute- Research Center
> 5000 East Belanger
> Montreal (Qc), Canada
> H1T 1C8
>
> 514-376-3330 ext 3511
> 514-376-1355 (Fax)
>
> [hidden email]

***************************
Tamara Howard
Cell Biology & Physiology
UNM-HSC
Albuquerque, NM
***************************

[Pertti Panula]

Hi,

In particular, with nestin and some other structural proteins, fixation
  may also be an issue. For a number of best available antibodies, PFA
is not optimal, but acetone works well. If needed, please contact prof.
Ismo Virtanen at ismo.virtanen at helsinki.fi for details.

Best regards Pertti



[hidden email] wrote:

>
> Bonjour à tous,
>
> We are trying to (indirect) immunostain with antiboby on  heart
> cryosections (14um).  The proteins that we want to identified are from
> the cytoskeleton like desmin, myosin light chain, myosin heavy chain.
>  We have a problem that all the staining stay on top of the tissue for
> those antibodies.  Counterstaining for actin (phalloidin alexa
> conjugated) is pretty good through the thickness of the tissue.  We
> permealized the tissue with Triton 0.5% , in the blocking solution
> (serum from the host of the 2nd Ab), for 1 hour and we incubate the
> primary overnight at  4C (with Triton 0.2% in the antibody diluent).
>  Other antibodies against cytoskeleton protein (myosin light chain 7)
> are well stained over the thickness of the tissue using the same
> protocol and the same batch of tissues.
>
> Any clue that might help us?
>
> Louissssssss
> Louis Villeneuve
> Research Associate- Confocal Microscopy
> Heart Montreal Institute- Research Center
> 5000 East Belanger
> Montreal (Qc), Canada
> H1T 1C8
>
> 514-376-3330 ext 3511
> 514-376-1355 (Fax)
>
> [hidden email]

--
Pertti Panula
Professor, Vice Dean of Research
Neuroscience Center
Institute of Biomedicine/Anatomy
Faculty of Medicine
POB 63, 00014 University of Helsinki
Finland
Phone: [phone]
Fax: [phone]
Mobile: [phone]
pertti.panula at helsinki.fi
http://www.helsinki.fi/neurosci/panula.htm

[Louis Villeneuve]

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Bonjour à tous,

I have a tiff image, 1392 x 1040 - 4.65 um square pixels acquired with a
camera interline Sony , 1.4 megapixel, color(7.6mm x 6.2mm array).  I use
a 40 x objective mounted on  a table microscope Leica DME.

Can I find the pixel size in hte image?

Thanks ,

Louis

[lechristophe]

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If there is no other magnifying/demagnifying optics in your
microscope, the theoretical pixel size is 4.65/40=116.25 nm

Christophe

On Wed, Oct 13, 2010 at 20:43,  <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Bonjour à tous,
>
> I have a tiff image, 1392 x 1040 - 4.65 um square pixels acquired with a
> camera interline Sony , 1.4 megapixel, color(7.6mm x 6.2mm array).  I use
> a 40 x objective mounted on  a table microscope Leica DME.
>
> Can I find the pixel size in hte image?
>
> Thanks ,
>
> Louis
>

[cromey]

*****
To join, leave or search the confocal microscopy listserv, go to:
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*****

Capture an image of a stage micrometer and you will know for sure.
Everything else is just educated guessing.
Doug

^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^
Douglas W. Cromey, M.S. - Assistant Scientific Investigator
Dept. of Cell Biology & Anatomy, University of Arizona
1501 N. Campbell Ave, Tucson, AZ  85724-5044 USA

office:  AHSC 4212         email: [hidden email]
voice:  520-626-2824       fax:  [phone]

http://swehsc.pharmacy.arizona.edu/exppath/
Home of: "Microscopy and Imaging Resources on the WWW"


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of Christophe Leterrier
Sent: Wednesday, October 13, 2010 12:04 PM
To: [hidden email]
Subject: Re: Pixel size

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

If there is no other magnifying/demagnifying optics in your
microscope, the theoretical pixel size is 4.65/40=116.25 nm

Christophe

On Wed, Oct 13, 2010 at 20:43,  <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Bonjour à tous,
>
> I have a tiff image, 1392 x 1040 - 4.65 um square pixels acquired with a
> camera interline Sony , 1.4 megapixel, color(7.6mm x 6.2mm array).  I use
> a 40 x objective mounted on  a table microscope Leica DME.
>
> Can I find the pixel size in hte image?
>
> Thanks ,
>
> Louis
>

[mmodel]

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To join, leave or search the confocal microscopy listserv, go to:
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I think most people get the pixel size from an image of a stage micrometer

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Wednesday, October 13, 2010 3:04 PM
To: [hidden email]
Subject: Re: Pixel size

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

If there is no other magnifying/demagnifying optics in your
microscope, the theoretical pixel size is 4.65/40=116.25 nm

Christophe

On Wed, Oct 13, 2010 at 20:43,  <[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Bonjour à tous,
>
> I have a tiff image, 1392 x 1040 - 4.65 um square pixels acquired with a
> camera interline Sony , 1.4 megapixel, color(7.6mm x 6.2mm array).  I use
> a 40 x objective mounted on  a table microscope Leica DME.
>
> Can I find the pixel size in hte image?
>
> Thanks ,
>
> Louis
>

[Cameron Nowell]

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Ok take two. First reply didn't go to the whole list.

For those that don't have access to a stage micrometer you can use a haemocytometer instead. The boxes on it ate 250, 200 and 50um.

Cheers

Cam


This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.

[Johannes Helm]

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Bonsoir, Louis,

that's one of the frequent questions I am being asked by "my" users.

The thing is:
Your microscope objective and condenser will provide a certain resolution,
officially dependent solely on numerical apertures. In case of a
transmitted light image, it will be dependent on the numerical apertures
of both, the condenser and the objective (proper Koehler illumination
assumed). The LATERAL microscope resolution in this case is

d = (1.2 * lambda) / (NA obj + NA cond.)

(at which, in case of day light filter operation, lambda is a somewhat
undefined thing, assume 500nm and / or use a panchromatic green filter to
reduce wavelength bandwidth).

In case of epi-illumination, the objective will also be the condenser, so
that the denominator reduces to (2*NA obj) though, in some special cases,
you might nevertheless end up with different ray paths for the
illumination and the detection, anyway, since the illumination ray path in
very special cases is a light cylinder around the "real" objective"
mirrored onto the preparation (annular illumination).

("Axial resolution" resp. "depth of field" in the wide field case is a
complicated and sometimes "debated" issue.)

You might call "d" the size of a "resel" (resolution element). Which is
different from a "pixel" (picture element).

Multiply the size of that resel with the magnification factor of all the
optics between the object and the chip of the camera. If you are lucky,
you have an adjustable zoom optics which allows you to adjust the
magnfication so that the image on the chip is magnified by a factor, which
makes sure the Nyquist theorem is fulfilled, at least. So: One resel
imaged onto the chip covers at least 2 pixels of the chip, preferrably
more (but not too much so that you do not oversample too much, loosing a
lot of light).

There is, in other words, to my mind not a straight forward answer to your
question. Unless you simply like to divide the dimensions of your camera
ship, which you can find in the manual of that camera, by the total
magnification factor. Then, you have the pixel size in the image, although
this might not really help you unless you compare it to the resel size as
mentioned above.

Best wishes,

Johannes

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Bonjour à tous,
>
> I have a tiff image, 1392 x 1040 - 4.65 um square pixels acquired with a
> camera interline Sony , 1.4 megapixel, color(7.6mm x 6.2mm array).  I use
> a 40 x objective mounted on  a table microscope Leica DME.
>
> Can I find the pixel size in hte image?
>
> Thanks ,
>
> Louis
>

--
P. Johannes Helm, M.Sc. PhD
Seniorengineer
CMBN
University of Oslo
Institute of Basic Medical Science
Department of Anatomy
Postboks 1105 - Blindern
NO-0317 Oslo

Voice: [phone]
Fax: [phone]

WWW: folk.uio.no/jhelm

[G. Esteban Fernandez]

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If there is no standard of known size available an alternative could
be to acquire images of any specimen before and after moving the stage
a known distance, if equipped with that capability.

-Esteban


On Wed, Oct 13, 2010 at 1:42 PM, Cameron Nowell
<[hidden email]> wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Ok take two. First reply didn't go to the whole list.
>
> For those that don't have access to a stage micrometer you can use a haemocytometer instead. The boxes on it ate 250, 200 and 50um.
>
> Cheers
>
> Cam
>
>
> This communication is intended only for the named recipient and may contain information that is confidential, legally privileged or subject to copyright; the Ludwig Institute for Cancer Research Ltd does not waive any rights if you have received this communication in error.
> The views expressed in this communication are those of the sender and do not necessarily reflect the views of the Ludwig Institute for Cancer Research Ltd.
>

[Andreas Bruckbauer]

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The problem is that the magnification of microscope objectives can vary a bit from one to another (same make and type). According to one manufacturer this can be +/- 5% for objectives with correction collar and +/- 2% without. Best to measure it but the theoretical pixel size is a good guess if you are sure about the additional magnification optics.
 
best wishes

Andreas


 

 

-----Original Message-----
From: Christophe Leterrier <[hidden email]>
To: [hidden email]
Sent: Wed, 13 Oct 2010 20:03
Subject: Re: Pixel size


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If there is no other magnifying/demagnifying optics in your

microscope, the theoretical pixel size is 4.65/40=116.25 nm



Christophe



On Wed, Oct 13, 2010 at 20:43,  <[hidden email]> wrote:

> *****

> To join, leave or search the confocal microscopy listserv, go to:

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

> *****

>

> Bonjour à tous,

>

> I have a tiff image, 1392 x 1040 - 4.65 um square pixels acquired with a

> camera interline Sony , 1.4 megapixel, color(7.6mm x 6.2mm array).  I use

> a 40 x objective mounted on  a table microscope Leica DME.

>

> Can I find the pixel size in hte image?

>

> Thanks ,

>

> Louis

>


 

[Mark Cannell]

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Hi All,

I think one should pay attention to the fact that this is a color  
camera implying a Bayer filter/mask. This effectively increases  pixel  
_pitch_  by a factor of 2.  Please note that it is pixel pitch that is  
critical for sampling/resolution issues _not_ pixel size.  RGB output  
for each pixel is calculated from the Bayer mask by the support  
electronics and this can fool you as to the actual sampling that is  
taking place. Fortunately, in monochrome cameras the pitch is often  
the same as the pixel size, but this is not always the case -you need  
to read the chip specification carefully.

In any case, I completely agree with other posts here: One should  
calibrate/check the overall magnification with a known test object  
because overall system magnification may not be solely determined by  
the nominal magnifications of the objective and (possible) relay lens.

My 2c

Cheers Mark


On 14/10/2010, at 9:53 AM, P. Johannes Helm wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Bonsoir, Louis,
>
> that's one of the frequent questions I am being asked by "my" users.
>
> The thing is:
> Your microscope objective and condenser will provide a certain  
> resolution,
> officially dependent solely on numerical apertures. In case of a
> transmitted light image, it will be dependent on the numerical  
> apertures
> of both, the condenser and the objective (proper Koehler illumination
> assumed). The LATERAL microscope resolution in this case is
>
> d = (1.2 * lambda) / (NA obj + NA cond.)
>
> (at which, in case of day light filter operation, lambda is a somewhat
> undefined thing, assume 500nm and / or use a panchromatic green  
> filter to
> reduce wavelength bandwidth).
>
> In case of epi-illumination, the objective will also be the  
> condenser, so
> that the denominator reduces to (2*NA obj) though, in some special  
> cases,
> you might nevertheless end up with different ray paths for the
> illumination and the detection, anyway, since the illumination ray  
> path in
> very special cases is a light cylinder around the "real" objective"
> mirrored onto the preparation (annular illumination).
>
> ("Axial resolution" resp. "depth of field" in the wide field case is a
> complicated and sometimes "debated" issue.)
>
> You might call "d" the size of a "resel" (resolution element). Which  
> is
> different from a "pixel" (picture element).
>
> Multiply the size of that resel with the magnification factor of all  
> the
> optics between the object and the chip of the camera. If you are  
> lucky,
> you have an adjustable zoom optics which allows you to adjust the
> magnfication so that the image on the chip is magnified by a factor,  
> which
> makes sure the Nyquist theorem is fulfilled, at least. So: One resel
> imaged onto the chip covers at least 2 pixels of the chip, preferrably
> more (but not too much so that you do not oversample too much,  
> loosing a
> lot of light).
>
> There is, in other words, to my mind not a straight forward answer  
> to your
> question. Unless you simply like to divide the dimensions of your  
> camera
> ship, which you can find in the manual of that camera, by the total
> magnification factor. Then, you have the pixel size in the image,  
> although
> this might not really help you unless you compare it to the resel  
> size as
> mentioned above.
>
> Best wishes,
>
> Johannes
>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Bonjour à tous,
>>
>> I have a tiff image, 1392 x 1040 - 4.65 um square pixels acquired  
>> with a
>> camera interline Sony , 1.4 megapixel, color(7.6mm x 6.2mm array).  
>> I use
>> a 40 x objective mounted on  a table microscope Leica DME.
>>
>> Can I find the pixel size in hte image?
>>
>> Thanks ,
>>
>> Louis
>>
> --
> P. Johannes Helm, M.Sc. PhD
> Seniorengineer
> CMBN
> University of Oslo
> Institute of Basic Medical Science
> Department of Anatomy
> Postboks 1105 - Blindern
> NO-0317 Oslo
>
> Voice: [phone]
> Fax: [phone]
>
> WWW: folk.uio.no/jhelm

[John Oreopoulos]

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Dear confocal listserver,

I'm looking for cheap or free sources of cells that can be used for testing various fluorescent stains and/or microscopy instrumentation. I've had good success in the past with blood cells pricked from my finger with a finger lance and cheek cells scraped from my mouth. I'm wondering if any of you out there know of any other sources of cells for this same kind of purpose. Maybe urine samples? Anything from nature/outdoors (besides pond water)? Anything from the supermarket?

Looking forward to all your creative responses!

John Oreopoulos