Strange artifact in confocal Z-stack

[lechristophe]

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

Dear all,

One of my colleagues brought me a Z-stack today that has a circular
pattern superimposed on his sample. It is taken with a Leica SPE
confocal, pinhole at 1 Airy unit, I guess the Z step is about 0.2 µm.
If you go down the stack trough the cell toward the glass (cells on
glass coverslip observed on an upright microscope), you see a circular
ring halo appearing that lowers in radius up to a circular spot at the
center of the field. A picture is worth a thousand words, so please
check the problem here :

http://ftp.espci.fr/incoming/christo/Montage.jpg

I've seen reflections from the coverslip before as a high background
fluorescence appearing when you reach the glass surface, but I wonder
if this is reflection or a defect in the objective, or something else.
Sis anyone have a similar problem ?

Thanks a lot for your help,

Christophe



Christophe Leterrier

Postdoc
INSERM UMR641 Neurobiology of ionic channels
IFR Jean Roche - Mediterranee University
Marseille, France

[Guy Cox]

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

I'm sure it must be a reflection from something - either slide,
coverslip or an air bubble in the sample.  2 suggestions to
check this out:

1. move the spectral detection sliders further from the excitation
wavelength and see if that makes it fainter.
2.  try an XZ section which might reveal the source.

By the way, thanks for putting the pictures up - it's so
much easier to comment on pictures than a vague description!

                                                 Guy



Optical Imaging Techniques in Cell Biology
by Guy Cox    CRC Press / Taylor & Francis
    http://www.guycox.com/optical.htm
______________________________________________
Associate Professor Guy Cox, MA, DPhil(Oxon)
Electron Microscope Unit, Madsen Building F09,
University of Sydney, NSW 2006
______________________________________________
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Mobile [phone]
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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Thursday, 29 May 2008 6:30 PM
To: [hidden email]
Subject: Strange artifact in confocal Z-stack

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

Dear all,

One of my colleagues brought me a Z-stack today that has a circular pattern superimposed on his sample. It is taken with a Leica SPE confocal, pinhole at 1 Airy unit, I guess the Z step is about 0.2 µm.
If you go down the stack trough the cell toward the glass (cells on glass coverslip observed on an upright microscope), you see a circular ring halo appearing that lowers in radius up to a circular spot at the center of the field. A picture is worth a thousand words, so please check the problem here :

http://ftp.espci.fr/incoming/christo/Montage.jpg

I've seen reflections from the coverslip before as a high background fluorescence appearing when you reach the glass surface, but I wonder if this is reflection or a defect in the objective, or something else.
Sis anyone have a similar problem ?

Thanks a lot for your help,

Christophe



Christophe Leterrier

Postdoc
INSERM UMR641 Neurobiology of ionic channels IFR Jean Roche - Mediterranee University Marseille, France

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[Monique Vasseur]

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

We had something similar with a Nikon upright microscope, except that we could see a similar effect in brightfield images -when we were using the "without fluorescence filter" position of the filter cube turet.  This was due to a leak of light in the filter cube turet when there was nothing in the "without fluorescent filter" position.  As soon as we put an empty cube in place, no more halo appeared.

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
-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Christophe Leterrier
Envoyé : 29 mai 2008 04:30
À : [hidden email]
Objet : Strange artifact in confocal Z-stack

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

Dear all,

One of my colleagues brought me a Z-stack today that has a circular
pattern superimposed on his sample. It is taken with a Leica SPE
confocal, pinhole at 1 Airy unit, I guess the Z step is about 0.2 µm.
If you go down the stack trough the cell toward the glass (cells on
glass coverslip observed on an upright microscope), you see a circular
ring halo appearing that lowers in radius up to a circular spot at the
center of the field. A picture is worth a thousand words, so please
check the problem here :

http://ftp.espci.fr/incoming/christo/Montage.jpg

I've seen reflections from the coverslip before as a high background
fluorescence appearing when you reach the glass surface, but I wonder
if this is reflection or a defect in the objective, or something else.
Sis anyone have a similar problem ?

Thanks a lot for your help,

Christophe



Christophe Leterrier

Postdoc
INSERM UMR641 Neurobiology of ionic channels
IFR Jean Roche - Mediterranee University
Marseille, France

[Paul Rigby-2]

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

Christophe,
What objective was used? We often see a similar reflection pattern from the coverslip or slide when using non-Plan objectives that have a curved focal plane.

We also sometimes see a pattern like this when a small air bubble is present in the immersion or mounting medium. And the bubble always migrates to the exact centre of the field!

Guy's suggestions should rule out reflections as the source.

Cheers
Paul Rigby


Dr Paul Rigby
Senior Lecturer
Centre for Microscopy, Characterisation and Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley  WA  6009
Ph (61-8) 9346 2819
Fx (61-8) 9346 3469

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Thursday, 29 May 2008 4:30 PM
To: [hidden email]
Subject: Strange artifact in confocal Z-stack

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

Dear all,

One of my colleagues brought me a Z-stack today that has a circular
pattern superimposed on his sample. It is taken with a Leica SPE
confocal, pinhole at 1 Airy unit, I guess the Z step is about 0.2 µm.
If you go down the stack trough the cell toward the glass (cells on
glass coverslip observed on an upright microscope), you see a circular
ring halo appearing that lowers in radius up to a circular spot at the
center of the field. A picture is worth a thousand words, so please
check the problem here :

http://ftp.espci.fr/incoming/christo/Montage.jpg

I've seen reflections from the coverslip before as a high background
fluorescence appearing when you reach the glass surface, but I wonder
if this is reflection or a defect in the objective, or something else.
Sis anyone have a similar problem ?

Thanks a lot for your help,

Christophe



Christophe Leterrier

Postdoc
INSERM UMR641 Neurobiology of ionic channels
IFR Jean Roche - Mediterranee University
Marseille, France

[Sarah Kefayati]

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

Dear Christophe,

 

I would say it is the reflection of your coverglass as I have seen this artifact once when I used an unknown coverglass.

do the imaging with the different cover glass and check if it's still there. 

 

Sarah

On Thu, May 29, 2008 at 8:50 PM, Paul Rigby <[hidden email]> wrote:

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

Christophe,
What objective was used? We often see a similar reflection pattern from the coverslip or slide when using non-Plan objectives that have a curved focal plane.

We also sometimes see a pattern like this when a small air bubble is present in the immersion or mounting medium. And the bubble always migrates to the exact centre of the field!

Guy's suggestions should rule out reflections as the source.

Cheers
Paul Rigby


Dr Paul Rigby
Senior Lecturer
Centre for Microscopy, Characterisation and Analysis (M510)
The University of Western Australia
35 Stirling Highway
Crawley  WA  6009
Ph (61-8) 9346 2819
Fx (61-8) 9346 3469

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Thursday, 29 May 2008 4:30 PM
To: [hidden email]
Subject: Strange artifact in confocal Z-stack

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

Dear all,

One of my colleagues brought me a Z-stack today that has a circular
pattern superimposed on his sample. It is taken with a Leica SPE
confocal, pinhole at 1 Airy unit, I guess the Z step is about 0.2 µm.
If you go down the stack trough the cell toward the glass (cells on
glass coverslip observed on an upright microscope), you see a circular
ring halo appearing that lowers in radius up to a circular spot at the
center of the field. A picture is worth a thousand words, so please
check the problem here :

http://ftp.espci.fr/incoming/christo/Montage.jpg

I've seen reflections from the coverslip before as a high background
fluorescence appearing when you reach the glass surface, but I wonder
if this is reflection or a defect in the objective, or something else.
Sis anyone have a similar problem ?

Thanks a lot for your help,

Christophe



Christophe Leterrier

Postdoc
INSERM UMR641 Neurobiology of ionic channels
IFR Jean Roche - Mediterranee University
Marseille, France

[Stephen Cody]

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

D'day Christophe,

I think that it is non-specific fluorescence on the outside of your tissue section. Did you use an antibody label? If so it may represent non-specific binding to the outside of the section. If it was a lipophilic retrograde label such as DiI, these can wash out of the membrane when in glycerol based mountants, and it may have become trapped between the section and the coverglass.

The curved nature of the artefact is probably as Paul suggests, because of the use of a non plan objective. Further evidence that you may be looking at the outside of the tissue section is that there appears to be knife chatter marks visible in panel 5.

Cheers

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Victoria,      3050
Australia
Tel: [phone]    Fax: [phone]
email: [hidden email]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal

Tip: Learn how to receive reminders about you microscope booking:
www.ludwig.edu.au/confocal/Local/Booking_Hint.htm  

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Thursday, 29 May 2008 6:30 PM
To: [hidden email]
Subject: Strange artifact in confocal Z-stack

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

Dear all,

One of my colleagues brought me a Z-stack today that has a circular
pattern superimposed on his sample. It is taken with a Leica SPE
confocal, pinhole at 1 Airy unit, I guess the Z step is about 0.2 µm.
If you go down the stack trough the cell toward the glass (cells on
glass coverslip observed on an upright microscope), you see a circular
ring halo appearing that lowers in radius up to a circular spot at the
center of the field. A picture is worth a thousand words, so please
check the problem here :

http://ftp.espci.fr/incoming/christo/Montage.jpg

I've seen reflections from the coverslip before as a high background
fluorescence appearing when you reach the glass surface, but I wonder
if this is reflection or a defect in the objective, or something else.
Sis anyone have a similar problem ?

Thanks a lot for your help,

Christophe



Christophe Leterrier

Postdoc
INSERM UMR641 Neurobiology of ionic channels
IFR Jean Roche - Mediterranee University
Marseille, France


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 does not waiver 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.

[Monique Vasseur]

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

Dear all,

I just want to complete my first response saying that we could see the same pattern in bright field on a Nikon upright fluorescence microscope when we used a empty position in the widefield fluorescence filter cube turret.

When in confocal mode, no widefield fluorescence filter cube should be in place.  When we were looking in brigtfield that day, we also were without fluorescence filter cube in place but the mercury lamp was on and the mercury lamp shutter was not closed. As soon as we closed the fluorescence shutter, the pattern disappeared in brightfield.  That was how we found that we needed an empty cube in that position to avoid interference with the mercury lamp light reflection/leak in the light path.

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
-----Message d'origine-----
De : Confocal Microscopy List [mailto:[hidden email]] De la part de Stephen Cody
Envoyé : 2 juin 2008 20:07
À : [hidden email]
Objet : Re: Strange artifact in confocal Z-stack

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

D'day Christophe,

I think that it is non-specific fluorescence on the outside of your tissue section. Did you use an antibody label? If so it may represent non-specific binding to the outside of the section. If it was a lipophilic retrograde label such as DiI, these can wash out of the membrane when in glycerol based mountants, and it may have become trapped between the section and the coverglass.

The curved nature of the artefact is probably as Paul suggests, because of the use of a non plan objective. Further evidence that you may be looking at the outside of the tissue section is that there appears to be knife chatter marks visible in panel 5.

Cheers

Stephen H. Cody
Microscopy Manager
Central Resource for Advanced Microscopy
Ludwig Institute for Cancer Research
PO Box 2008 Royal Melbourne Hospital
Victoria,      3050
Australia
Tel: [phone]    Fax: [phone]
email: [hidden email]
www.ludwig.edu.au/labs/confocal.html
www.ludwig.edu.au/confocal

Tip: Learn how to receive reminders about you microscope booking:
www.ludwig.edu.au/confocal/Local/Booking_Hint.htm  

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Christophe Leterrier
Sent: Thursday, 29 May 2008 6:30 PM
To: [hidden email]
Subject: Strange artifact in confocal Z-stack

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

Dear all,

One of my colleagues brought me a Z-stack today that has a circular
pattern superimposed on his sample. It is taken with a Leica SPE
confocal, pinhole at 1 Airy unit, I guess the Z step is about 0.2 µm.
If you go down the stack trough the cell toward the glass (cells on
glass coverslip observed on an upright microscope), you see a circular
ring halo appearing that lowers in radius up to a circular spot at the
center of the field. A picture is worth a thousand words, so please
check the problem here :

http://ftp.espci.fr/incoming/christo/Montage.jpg

I've seen reflections from the coverslip before as a high background
fluorescence appearing when you reach the glass surface, but I wonder
if this is reflection or a defect in the objective, or something else.
Sis anyone have a similar problem ?

Thanks a lot for your help,

Christophe



Christophe Leterrier

Postdoc
INSERM UMR641 Neurobiology of ionic channels
IFR Jean Roche - Mediterranee University
Marseille, France


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 does not waiver 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.

[John Oreopoulos]

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

I have an unusual situation that I'm hoping someone out there might be able to advise me on. I would like to modify my lab's inverted Olympus IX70 microscope to have the ability to look at polarized light when illuminated in the transmission pathway. I want to polarize the white light that illuminates the sample, and then analyze the polarization of the image by examining the light that comes through the sample with another polarizer that can be oriented parallel and perpendicular to the direction of the polarizer in front of the light source. 

We have DIC optics for the microscope - this means that I have a polarizer that can be fitted on top of the light source condenser, and I also have a fixed position analyzer that can slide in below the fluorescence filter cube turret. My problem is that the polarizer in the analyzer slider is fixed in a direction that is crossed to the polarizer in front of the light source. What I really want to do is to be able to rotate the the polarizer in the slider to the parallel direction as well. I know that Olympus and Leeds sells rotating analyzers that can slot into these positions on the microscope, but my supervisor does not have the funds to afford these right now. So I've come up with a slightly cheaper alternative solution.

We bought two 1" round film polarizers from Edmund optics (~20$ each). These are the exact same size as interference filters that normally get fitted into the fluorescence filter cubes which go in the rotating turret below the objective nosepiece on the inverted microscope. My plan was to take two empty fluorescence filter cubes and insert a polarizer in each - one oriented in the parallel direction and one in the perpendicular direction. Here is my problem: how can I orient and lock the position of the the two polarizers in the filter cubes such that they are aligned with their polarization axis directions correctly? Ideally, since I'm trying to be quantitative with the imaging, I'd like their alignment from their relative crossed position to be within 1 degree or less. When I screw them down with the retaining ring of the filter cube, they rotate slightly in the cube before they become locked. Also, I can't tell if I'm dropping them into the filter holder in the right position since their reference axis position is marked on the edge of the filters. Can anyone out there suggest a good method to overcome this problem? How do the microscope manufacturers align their polarizers in the sliders they sell for DIC imaging, etc?

Thank you in advance for any help!

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

Tel: W:416-946-5022

[Barbara Foster]

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

Your idea about putting the polarizer on top of the condenser is actually a better one than to have both in filter cubes.  I can't remember the exact configuration of the filter slot there, but I would check to see if it will give you enough room so that you could glue a small handle to your polarizer and still rotate it, at least through 45 degrees (90 would be ideal). 

As for the analyzer: a less elegant but perhaps more controllable approach might be to remove the binocular body and install it in that interface location, maybe with a small retaining ring to keep it in place.  Since the binoc fits on with a dovetail, that location might give you the ability to align without disturbing when you "tighten down". 

Re: alignment.  The ultimate test is full extinction when the two polarizers are crossed. In an optics lab, you can test those positions by shining light through the two into spectrometer.   Obviously, you need to calibrate one against a known first to learn its permitted direction of vibration, then rotate the second until the intensity of light emerging reaches a minimum. 

One other caveat:  you may want to test your objectives to see if they de-polarize the light.  Most of us in biology want high NA and high resolution and therefore would probably prefer to work with apochromats.  Unfortunately, there are typically on the order of 17-21 lenselts inside, each of which is subject to strain which will depolarize the illumination. There is also the issue of the glass chemistry.   Fluorites are often a better choice.  I would recommend that, when you are trying to find the fully crossed position for your two polars, you do so with no optics in the way.  That will, essentially, provide a bare system response.  Then you can rotate each objective into the path and observe the intensity of the background. If it goes from velvety black to charcoal gray, you know that the optics are depolarizing the light. 

By the way, there is a clever device that is about to come onto the market called a "QLS" (Quantitive Light Source") that might be helpful in the testing phase.  Contact James Beach" <[hidden email]> (I've cc'd him on this response). 

Good hunting!
Barbara

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At 12:57 PM 6/5/2008, you wrote:

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

I have an unusual situation that I'm hoping someone out there might be able to advise me on. I would like to modify my lab's inverted Olympus IX70 microscope to have the ability to look at polarized light when illuminated in the transmission pathway. I want to polarize the white light that illuminates the sample, and then analyze the polarization of the image by examining the light that comes through the sample with another polarizer that can be oriented parallel and perpendicular to the direction of the polarizer in front of the light source.
We have DIC optics for the microscope - this means that I have a polarizer that can be fitted on top of the light source condenser, and I also have a fixed position analyzer that can slide in below the fluorescence filter cube turret. My problem is that the polarizer in the analyzer slider is fixed in a direction that is crossed to the polarizer in front of the light source. What I really want to do is to be able to rotate the the polarizer in the slider to the parallel direction as well. I know that Olympus and Leeds sells rotating analyzers that can slot into these positions on the microscope, but my supervisor does not have the funds to afford these right now. So I've come up with a slightly cheaper alternative solution.
We bought two 1" round film polarizers from Edmund optics (~20$ each). These are the exact same size as interference filters that normally get fitted into the fluorescence filter cubes which go in the rotating turret below the objective nosepiece on the inverted microscope. My plan was to take two empty fluorescence filter cubes and insert a polarizer in each - one oriented in the parallel direction and one in the perpendicular direction. Here is my problem: how can I orient and lock the position of the the two polarizers in the filter cubes such that they are aligned with their polarization axis directions correctly? Ideally, since I'm trying to be quantitative with the imaging, I'd like their alignment from their relative crossed position to be within 1 degree or less. When I screw them down with the retaining ring of the filter cube, they rotate slightly in the cube before they become locked. Also, I can't tell if I'm dropping them into the filter holder in the right position since their reference axis position is marked on the edge of the filters. Can anyone out there suggest a good method to overcome this problem? How do the microscope manufacturers align their polarizers in the sliders they sell for DIC imaging, etc?

Thank you in advance for any help!


John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging


Tel: W:416-946-5022

[Haller, Edward]

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

John,

 

            Here are a few thoughts. First, try mounting a moving polarizer above your condenser and use your condenser in the transmitted light position. You would be able to use your fixed analyzer and then spin the polarizer to parallel or crossed position. This would be the simplest answer. Fashion some type of a holder for the polarizer and simply sit it on top of the condenser, knowing which direction the polarizer is oriented, you could quickly manually orient the filter for either position. Some microscopes come with empty, spare filter holders that fit between the light source and the condenser. Check to see if you have one, and then adapt your filter to the holder.

If you need to mount analyzers in your filter cubes, try the following:  Do you have access to a light box? If so, put a polarizing filter on top of the box. Adhere it to the box with some tape by the edges so it will not shift. Put your filter cube with polarizer in it above the first polarizer, and orient it either to parallel or crossed position. Use a small drop of nail polish on a toothpick at an edge of the polarizer to “glue” it in place in the filter cube, then carefully screw the retaining ring into place once the glue has hardened. This is a trick I’ve seen with some older optics when someone needed to orient a filter or lens in an exact position. I don’t know how reasonable it is to expect 1 degree accuracy, but I’m willing to bet that if the filters stay put when you screw in the retaining rings, you’ll have your solution. The key, obviously, is to be careful with the glue so it doesn’t ruin the filters. Superglue would be too low in viscosity, and hot melt glue from a glue gun might not hold securely, and is flexible. If you have glass polarizers, the nail polish can be removed from the filters with acetone or non-toxic polish remover if necessary for repositioning. The nail polish dries slowly enough that you would have time to turn the polarizer in the holder if you don’t align it perfectly to start with.

Another possibility would be to go to a camera store and to purchase a polarizing filter mounted to fit on a 35mm camera lens. These can’t be too expensive. You could use this lens above your condenser in place of a rotatable analyzer. Whether these suggestions solve your problem or not, please post your answer so the rest of us can benefit. Thank you, John.

 

Ed Haller

 

Edward Haller, Lab Manager

Microscopy Core Labs

University of South Florida

College of Medicine

Tampa, FL 33612

813-974-0569

 

---

From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Thursday, June 05, 2008 2:51 PM
To: [hidden email]
Subject: How to align polarization filters in two filter cubes

 

Hello confocal listserver,

 

I have an unusual situation that I'm hoping someone out there might be able to advise me on. I would like to modify my lab's inverted Olympus IX70 microscope to have the ability to look at polarized light when illuminated in the transmission pathway. I want to polarize the white light that illuminates the sample, and then analyze the polarization of the image by examining the light that comes through the sample with another polarizer that can be oriented parallel and perpendicular to the direction of the polarizer in front of the light source. 

We have DIC optics for the microscope - this means that I have a polarizer that can be fitted on top of the light source condenser, and I also have a fixed position analyzer that can slide in below the fluorescence filter cube turret. My problem is that the polarizer in the analyzer slider is fixed in a direction that is crossed to the polarizer in front of the light source. What I really want to do is to be able to rotate the the polarizer in the slider to the parallel direction as well. I know that Olympus and Leeds sells rotating analyzers that can slot into these positions on the microscope, but my supervisor does not have the funds to afford these right now. So I've come up with a slightly cheaper alternative solution.

We bought two 1" round film polarizers from Edmund optics (~20$ each). These are the exact same size as interference filters that normally get fitted into the fluorescence filter cubes which go in the rotating turret below the objective nosepiece on the inverted microscope. My plan was to take two empty fluorescence filter cubes and insert a polarizer in each - one oriented in the parallel direction and one in the perpendicular direction. Here is my problem: how can I orient and lock the position of the the two polarizers in the filter cubes such that they are aligned with their polarization axis directions correctly? Ideally, since I'm trying to be quantitative with the imaging, I'd like their alignment from their relative crossed position to be within 1 degree or less. When I screw them down with the retaining ring of the filter cube, they rotate slightly in the cube before they become locked. Also, I can't tell if I'm dropping them into the filter holder in the right position since their reference axis position is marked on the edge of the filters. Can anyone out there suggest a good method to overcome this problem? How do the microscope manufacturers align their polarizers in the sliders they sell for DIC imaging, etc?

 

Thank you in advance for any help!

 

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

 

Tel: W:416-946-5022

 

[John Oreopoulos]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Thanks Ed and Barbara. I'm going to try Ed's nail-polish idea.

John

On 5-Jun-08, at 3:42 PM, Haller, Edward wrote:

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

John,

 

            Here are a few thoughts. First, try mounting a moving polarizer above your condenser and use your condenser in the transmitted light position. You would be able to use your fixed analyzer and then spin the polarizer to parallel or crossed position. This would be the simplest answer. Fashion some type of a holder for the polarizer and simply sit it on top of the condenser, knowing which direction the polarizer is oriented, you could quickly manually orient the filter for either position. Some microscopes come with empty, spare filter holders that fit between the light source and the condenser. Check to see if you have one, and then adapt your filter to the holder.

If you need to mount analyzers in your filter cubes, try the following:  Do you have access to a light box? If so, put a polarizing filter on top of the box. Adhere it to the box with some tape by the edges so it will not shift. Put your filter cube with polarizer in it above the first polarizer, and orient it either to parallel or crossed position. Use a small drop of nail polish on a toothpick at an edge of the polarizer to “glue” it in place in the filter cube, then carefully screw the retaining ring into place once the glue has hardened. This is a trick I’ve seen with some older optics when someone needed to orient a filter or lens in an exact position. I don’t know how reasonable it is to expect 1 degree accuracy, but I’m willing to bet that if the filters stay put when you screw in the retaining rings, you’ll have your solution. The key, obviously, is to be careful with the glue so it doesn’t ruin the filters. Superglue would be too low in viscosity, and hot melt glue from a glue gun might not hold securely, and is flexible. If you have glass polarizers, the nail polish can be removed from the filters with acetone or non-toxic polish remover if necessary for repositioning. The nail polish dries slowly enough that you would have time to turn the polarizer in the holder if you don’t align it perfectly to start with.

Another possibility would be to go to a camera store and to purchase a polarizing filter mounted to fit on a 35mm camera lens. These can’t be too expensive. You could use this lens above your condenser in place of a rotatable analyzer. Whether these suggestions solve your problem or not, please post your answer so the rest of us can benefit. Thank you, John.

 

Ed Haller

 

Edward Haller, Lab Manager

Microscopy Core Labs

University of South Florida

College of Medicine

Tampa, FL 33612

813-974-0569

 

---

From: Confocal Microscopy List [[hidden email]] On Behalf Of John Oreopoulos
Sent: Thursday, June 05, 2008 2:51 PM
To: [hidden email]
Subject: How to align polarization filters in two filter cubes

 

Hello confocal listserver,

 

I have an unusual situation that I'm hoping someone out there might be able to advise me on. I would like to modify my lab's inverted Olympus IX70 microscope to have the ability to look at polarized light when illuminated in the transmission pathway. I want to polarize the white light that illuminates the sample, and then analyze the polarization of the image by examining the light that comes through the sample with another polarizer that can be oriented parallel and perpendicular to the direction of the polarizer in front of the light source. 

We have DIC optics for the microscope - this means that I have a polarizer that can be fitted on top of the light source condenser, and I also have a fixed position analyzer that can slide in below the fluorescence filter cube turret. My problem is that the polarizer in the analyzer slider is fixed in a direction that is crossed to the polarizer in front of the light source. What I really want to do is to be able to rotate the the polarizer in the slider to the parallel direction as well. I know that Olympus and Leeds sells rotating analyzers that can slot into these positions on the microscope, but my supervisor does not have the funds to afford these right now. So I've come up with a slightly cheaper alternative solution.

We bought two 1" round film polarizers from Edmund optics (~20$ each). These are the exact same size as interference filters that normally get fitted into the fluorescence filter cubes which go in the rotating turret below the objective nosepiece on the inverted microscope. My plan was to take two empty fluorescence filter cubes and insert a polarizer in each - one oriented in the parallel direction and one in the perpendicular direction. Here is my problem: how can I orient and lock the position of the the two polarizers in the filter cubes such that they are aligned with their polarization axis directions correctly? Ideally, since I'm trying to be quantitative with the imaging, I'd like their alignment from their relative crossed position to be within 1 degree or less. When I screw them down with the retaining ring of the filter cube, they rotate slightly in the cube before they become locked. Also, I can't tell if I'm dropping them into the filter holder in the right position since their reference axis position is marked on the edge of the filters. Can anyone out there suggest a good method to overcome this problem? How do the microscope manufacturers align their polarizers in the sliders they sell for DIC imaging, etc?

 

Thank you in advance for any help!

 

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging

 

Tel: W:416-946-5022

 

[John Oreopoulos]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal I've been playing around with it a bit, and I've discovered that an LCD computer monitor makes a very good polarized "light box" that can be used to check the orientation of the filter once it's in the filter cube. I should pass on though that not all LCD screens emit polarized light in the same direction; one of our lab computer screens is horizontally polarized while my PowerBook Mac laptop screen is polarized at 45 degrees from the vertical.

John

On 5-Jun-08, at 3:36 PM, Barbara Foster wrote:

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

Your idea about putting the polarizer on top of the condenser is actually a better one than to have both in filter cubes.  I can't remember the exact configuration of the filter slot there, but I would check to see if it will give you enough room so that you could glue a small handle to your polarizer and still rotate it, at least through 45 degrees (90 would be ideal). 

As for the analyzer: a less elegant but perhaps more controllable approach might be to remove the binocular body and install it in that interface location, maybe with a small retaining ring to keep it in place.  Since the binoc fits on with a dovetail, that location might give you the ability to align without disturbing when you "tighten down". 

Re: alignment.  The ultimate test is full extinction when the two polarizers are crossed. In an optics lab, you can test those positions by shining light through the two into spectrometer.   Obviously, you need to calibrate one against a known first to learn its permitted direction of vibration, then rotate the second until the intensity of light emerging reaches a minimum. 

One other caveat:  you may want to test your objectives to see if they de-polarize the light.  Most of us in biology want high NA and high resolution and therefore would probably prefer to work with apochromats.  Unfortunately, there are typically on the order of 17-21 lenselts inside, each of which is subject to strain which will depolarize the illumination. There is also the issue of the glass chemistry.   Fluorites are often a better choice.  I would recommend that, when you are trying to find the fully crossed position for your two polars, you do so with no optics in the way.  That will, essentially, provide a bare system response.  Then you can rotate each objective into the path and observe the intensity of the background. If it goes from velvety black to charcoal gray, you know that the optics are depolarizing the light. 

By the way, there is a clever device that is about to come onto the market called a "QLS" (Quantitive Light Source") that might be helpful in the testing phase.  Contact James Beach" <[hidden email]> (I've cc'd him on this response). 

Good hunting!
Barbara

Barbara Foster, President

Microscopy/Microscopy Education
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McKinney TX 75070
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MME is now scheduling customized, on-site courses through Sept 2008.  Call us today for details.



At 12:57 PM 6/5/2008, you wrote:

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

I have an unusual situation that I'm hoping someone out there might be able to advise me on. I would like to modify my lab's inverted Olympus IX70 microscope to have the ability to look at polarized light when illuminated in the transmission pathway. I want to polarize the white light that illuminates the sample, and then analyze the polarization of the image by examining the light that comes through the sample with another polarizer that can be oriented parallel and perpendicular to the direction of the polarizer in front of the light source.
We have DIC optics for the microscope - this means that I have a polarizer that can be fitted on top of the light source condenser, and I also have a fixed position analyzer that can slide in below the fluorescence filter cube turret. My problem is that the polarizer in the analyzer slider is fixed in a direction that is crossed to the polarizer in front of the light source. What I really want to do is to be able to rotate the the polarizer in the slider to the parallel direction as well. I know that Olympus and Leeds sells rotating analyzers that can slot into these positions on the microscope, but my supervisor does not have the funds to afford these right now. So I've come up with a slightly cheaper alternative solution.
We bought two 1" round film polarizers from Edmund optics (~20$ each). These are the exact same size as interference filters that normally get fitted into the fluorescence filter cubes which go in the rotating turret below the objective nosepiece on the inverted microscope. My plan was to take two empty fluorescence filter cubes and insert a polarizer in each - one oriented in the parallel direction and one in the perpendicular direction. Here is my problem: how can I orient and lock the position of the the two polarizers in the filter cubes such that they are aligned with their polarization axis directions correctly? Ideally, since I'm trying to be quantitative with the imaging, I'd like their alignment from their relative crossed position to be within 1 degree or less. When I screw them down with the retaining ring of the filter cube, they rotate slightly in the cube before they become locked. Also, I can't tell if I'm dropping them into the filter holder in the right position since their reference axis position is marked on the edge of the filters. Can anyone out there suggest a good method to overcome this problem? How do the microscope manufacturers align their polarizers in the sliders they sell for DIC imaging, etc?

Thank you in advance for any help!


John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging


Tel: W:416-946-5022

[ChrisWilms]

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

Barbara correctly pointed to the role of objectives. But the situation  
is worse: even perfectly strainless lenses will depolarize light. The  
process of focusing light causes this depolarization and it becomes a  
significant factor when using high NA objectives. For some literature  
on this you might want to have a look at:

Methods Cell Biology 1989;30:333-52.
"Fluorescence polarization microscopy."
Axelrod, D.

Biophysical Journal, Vol 26, 557-573,
"Carbocyanine dye orientation in red cell membrane studied by  
microscopic fluorescence polarization"
Axelrod, D.

Regards, Christian


--
Christian Wilms
Wolfson Institute for Biomedical Research
University College London
Gower Street
London, WC1E 6BT
United Kingdom

[Gustin, Emmanuel [TIBBE]]

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

For your attention, on behalf of my colleague Eberhard and myself:

Dear colleagues,

We have currently an open Image Analyst position to support our cell biologists in their efforts in drug discovery and to assist particularly in assay development for high-content screening applying automated high-throughput microscopy. For a detailed job description please follow the link (or see below):

http://www.tibotec.com/bgdisplay.jhtml?itemname=stars

Perhaps you are personally interested. Otherwise, could you please spread the information among your networks, and please post if possible a print of the job advertisement at appropriate places of your institutions.

Thank you very much for your valuable support in advance.

Best regards,

Eberhard Krauß


The Position:
Image Analyst for High Content Screening-

Description
* You support assay development and data analysis in drug discovery research.
Furthermore, you develop image analysis protocols for high-content screening (HCS) platforms.
* You work in a team with cell biologists and virologists to develop new cell-based assays on an automated microscopy platform, and advise researchers on strategies to achieve the best results.
* You take custody of the data flow throughout the HCS process, ensuring excellent data quality.
* You work closely together with the system users and the IT department to provide suitable tools for HCS data analysis and reporting.
* You report to the Director of Discovery Assay Technologies.

Qualifications
* You should have a post graduate qualification in science or engineering. Strong data analysis and programming skills, a good understanding of experimental methods are essential.
* You should have basic understanding of the fundamentals of cell biology and microscopy.
* Experience in high content screening and image analysis is advantageous.
* This position calls for a person with a strong interest in complex data analysis and data quality, who can contribute effectively to a multi-disciplinary team. Good communication skills are required.

Please note that this position is open to both young and experienced colleagues, as well as to both Belgian and non-Belgian candidates.

Job RD-General
Primary LocationBE-VAN-Mechelen
Schedule Full-time
Job Type Standard
Tibotec BVBA

_________________________________________________

Dr. Eberhard Krausz
Director, Discovery Assay Technologies (DAT)
[hidden email]
Tel.: [phone]
FAX : [phone]

Emmanuel Gustin, PhD  
Principal Scientist
[hidden email]
Tel.: [phone]
Tel.: [phone]

Tibotec bvba
Generaal De Wittelaan L11 B3
B-2800 Mechelen
Belgium

[John Oreopoulos]

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

I don't normally advertise articles on the confocal list server, but I think that this one was particularly well written and I know that there is a lot of interest in this topic, so I thought I'd let everyone know about it. Here is an up-to-date physicsworld article on the work being done to break the diffraction limit in optical microscopy:

http://physicsworld.com/cws/article/print/34322;jsessionid=CE7FEA2A307D82032AD91613FBDDB0E2

Enjoy!

John Oreopoulos, BSc,

PhD Candidate

University of Toronto

Institute For Biomaterials and Biomedical Engineering

Centre For Studies in Molecular Imaging