Amount of shear needed for DIC imaging (and provided by Zeiss objectives)

[Shalin Mehta]

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

What does the shear required by DIC depend on? Magnification or NA? To me it appears that only magnification should matter as DIC measures the phase gradient in direction of shear between two points in space. Any good reference that quantitatively relates magnification/NA to the shear required?

I wish to do time-lapse of live cells in DIC. I have a 40x 0.75 NA and 40x 1.3 NA oil objectives from Zeiss. For the 0.75 NA objective we do not have DIC prism but for 1.3NA we have (as per Zeiss nomencleture DIC III). During time-lapse I see lot of focal drift with oil objective but with air objective focus is relatively stable. Is it alright to use 1.3 NA objective's prism with 0.75 NA objective?

I have taken some images with such configuration and I think the match is not perfect - because when changing the bias retardation by moving prism I do not see transition from positive gradient to zero contrast to negative gradient.

Thanks for all inputs

Best regards
Shalin

--
My co-ordinates:
Shalin Mehta, Graduate student
Graduate Programme in Bioengineering, NUS, Singapore
Email: shalin {dot} mehta {at} gmail {dot} com
Blog: <a href="http://electricsbm.blogspot.com" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">electricsbm.blogspot.com
Mobile: [phone]

[Barbara Foster]

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

The shear is actually defined by the prisms in the system and it tied to the NA.

Set up DIC then remove the eyepiece and peer down the tube into the back focal plane of the objective.  If everything is set correctly, you should see a white background with a dark fringe in down the middle.  This is an interference fringe, derived from the shear between what can be thought of the real image and the reference image. That shear is created by the prism.  The reason that you don't see a ghost image in conventional DIC (which you WOULD see in other, related techniques, such as Jamin-Lebedeff), is that the shear is smaller than the resolving power of the objective.  Hence the name "differential" (small).

For the DIC background to be even, the fringe pretty much needs to fill most of the back focal plane, so the prism is engineered to fit the NA. 

Since your 40x dry is lower in NA than your oil objective, I would go ahead try the prism. You may have to adjust the compensator a bit further, but it is worth the try.  If one of the Zeiss specialists is "lurking," perhaps they can give you a more definitive answer.

I wrote a comprehensive "lay" article on DIC in American Lab, April 1988 that you might find helpful.  We've tried scanning it but have had problems. I am on the road through 10Sept but can try again after that, if you are interested.  There is also a detailed description in "Optimizing Light Microscopy," and I suspect that there will be dynamic Java tutorials on both the Olympus micro site and the Nikon uUniversity. 

By the way, because of all the mis-interpretation that comes from features in the field that respond to polarized light, I modified my instructions for DIC set up some years ago. The "Foster Method" :
1. Establish Koehler and observe any native color
2. Cross polars and observe any features that are bright against the dark background.  They will respond to the polarizing part of the system rather than the DIC beam shearing part.
3. Insert the DIC prisms (usually in the nosepiece and the MATCHING prism in the condenser)
4. Tune the compensator to soft dove gray background (the article explains why this is the most sensitive position).

Hope this is helpful.  Good hunting!
Barbara Foster, President

We've moved!
Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: [phone]
Skype: fostermme
W: www.MicroscopyEducation.com


MME is now scheduling customized, on-site courses through December.  Call us today for details.

P. S.
Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy".  Copies still available through MME... even for class-room lots ... and we give quantity discounts. Just call us here in the MME office for details.







At 09:08 PM 8/26/2007, you wrote:

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

What does the shear required by DIC depend on? Magnification or NA? To me it appears that only magnification should matter as DIC measures the phase gradient in direction of shear between two points in space. Any good reference that quantitatively relates magnification/NA to the shear required?

I wish to do time-lapse of live cells in DIC. I have a 40x 0.75 NA and 40x 1.3 NA oil objectives from Zeiss. For the 0.75 NA objective we do not have DIC prism but for 1.3NA we have (as per Zeiss nomencleture DIC III). During time-lapse I see lot of focal drift with oil objective but with air objective focus is relatively stable. Is it alright to use 1.3 NA objective's prism with 0.75 NA objective?

I have taken some images with such configuration and I think the match is not perfect - because when changing the bias retardation by moving prism I do not see transition from positive gradient to zero contrast to negative gradient.

Thanks for all inputs

Best regards
Shalin

--
My co-ordinates:
Shalin Mehta, Graduate student
Graduate Programme in Bioengineering, NUS, Singapore
Email: shalin {dot} mehta {at} gmail {dot} com
Blog: electricsbm.blogspot.com
Mobile: [phone]

[Shalin Mehta]

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

 Thanks for all the tips. Sorry I didn't catch what you meant by observing native color with Kohler illumination. I will indeed like to have the article. Unfortunately, our library doesn't subscribe to American lab magazine. Please send it to me once you are back in office.

Recently Prof. Colin mentioned to me that compensator setting that will keep all the phase information just positive in image(i.e. the lowest phase in object within field of view is mapped to just close to zero amplitude in image) gives the highest contrast. He has published this in a paper on reflection DIC. I suppose this is the soft-dove background you referred to.

Many thanks
Regards
Shalin

On 8/27/07, Barbara Foster <[hidden email]> wrote:

Search the CONFOCAL archive at <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal

Hi, Shalin

The shear is actually defined by the prisms in the system and it tied to the NA.

Set up DIC then remove the eyepiece and peer down the tube into the back focal plane of the objective.  If everything is set correctly, you should see a white background with a dark fringe in down the middle.  This is an interference fringe, derived from the shear between what can be thought of the real image and the reference image. That shear is created by the prism.  The reason that you don't see a ghost image in conventional DIC (which you WOULD see in other, related techniques, such as Jamin-Lebedeff), is that the shear is smaller than the resolving power of the objective.  Hence the name "differential" (small).

For the DIC background to be even, the fringe pretty much needs to fill most of the back focal plane, so the prism is engineered to fit the NA. 

Since your 40x dry is lower in NA than your oil objective, I would go ahead try the prism. You may have to adjust the compensator a bit further, but it is worth the try.  If one of the Zeiss specialists is "lurking," perhaps they can give you a more definitive answer.

I wrote a comprehensive "lay" article on DIC in American Lab, April 1988 that you might find helpful.  We've tried scanning it but have had problems. I am on the road through 10Sept but can try again after that, if you are interested.  There is also a detailed description in "Optimizing Light Microscopy," and I suspect that there will be dynamic Java tutorials on both the Olympus micro site and the Nikon uUniversity. 

By the way, because of all the mis-interpretation that comes from features in the field that respond to polarized light, I modified my instructions for DIC set up some years ago. The "Foster Method" :
1. Establish Koehler and observe any native color
2. Cross polars and observe any features that are bright against the dark background.  They will respond to the polarizing part of the system rather than the DIC beam shearing part.
3. Insert the DIC prisms (usually in the nosepiece and the MATCHING prism in the condenser)
4. Tune the compensator to soft dove gray background (the article explains why this is the most sensitive position).

Hope this is helpful.  Good hunting!
Barbara Foster, President

We've moved!
Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: [phone]
Skype: fostermme
W: <a href="http://www.microscopyeducation.com/" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> www.MicroscopyEducation.com


MME is now scheduling customized, on-site courses through December.  Call us today for details.

P. S.
Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy".  Copies still available through MME... even for class-room lots ... and we give quantity discounts. Just call us here in the MME office for details.







At 09:08 PM 8/26/2007, you wrote:

Search the CONFOCAL archive at <a href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Dear List,

What does the shear required by DIC depend on? Magnification or NA? To me it appears that only magnification should matter as DIC measures the phase gradient in direction of shear between two points in space. Any good reference that quantitatively relates magnification/NA to the shear required?

I wish to do time-lapse of live cells in DIC. I have a 40x 0.75 NA and 40x 1.3 NA oil objectives from Zeiss. For the 0.75 NA objective we do not have DIC prism but for 1.3NA we have (as per Zeiss nomencleture DIC III). During time-lapse I see lot of focal drift with oil objective but with air objective focus is relatively stable. Is it alright to use 1.3 NA objective's prism with 0.75 NA objective?

I have taken some images with such configuration and I think the match is not perfect - because when changing the bias retardation by moving prism I do not see transition from positive gradient to zero contrast to negative gradient.

Thanks for all inputs

Best regards
Shalin

--
My co-ordinates:
Shalin Mehta, Graduate student
Graduate Programme in Bioengineering, NUS, Singapore
Email: shalin {dot} mehta {at} gmail {dot} com
Blog: <a href="http://electricsbm.blogspot.com" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)">electricsbm.blogspot.com
Mobile: [phone]

--
My co-ordinates:
Shalin Mehta, Graduate student
Graduate Programme in Bioengineering, NUS, Singapore
Email: shalin {dot} mehta {at} gmail {dot} com
Blog: <a href="http://electricsbm.blogspot.com" target="_blank" onclick="return top.js.OpenExtLink(window,event,this)"> electricsbm.blogspot.com
Mobile: [phone]

[Barbara Foster]

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

Native color just refers to the fact that some entities have color of their own that can affect what you see under polarized light or DIC.

Re: the setting - sounds like you have the right location (soft dove gray).

Best regards,
Barbara Foster, President

We've moved!
Microscopy/Microscopy Education
7101 Royal Glen Trail, Suite A
McKinney TX 75070
P: [phone]
Skype: fostermme
W: www.MicroscopyEducation.com


MME is now scheduling customized, on-site courses through December.  Call us today for details.

P. S.
Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy".  Copies still available through MME... even for class-room lots ... and we give quantity discounts. Just call us here in the MME office for details.



At 08:01 PM 8/29/2007, you wrote:

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

 Thanks for all the tips. Sorry I didn't catch what you meant by observing native color with Kohler illumination. I will indeed like to have the article. Unfortunately, our library doesn't subscribe to American lab magazine. Please send it to me once you are back in office.

Recently Prof. Colin mentioned to me that compensator setting that will keep all the phase information just positive in image(i.e. the lowest phase in object within field of view is mapped to just close to zero amplitude in image) gives the highest contrast. He has published this in a paper on reflection DIC. I suppose this is the soft-dove background you referred to.

Many thanks
Regards
Shalin

On 8/27/07, Barbara Foster <[hidden email]> wrote:

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

Hi, Shalin

The shear is actually defined by the prisms in the system and it tied to the NA.

Set up DIC then remove the eyepiece and peer down the tube into the back focal plane of the objective.  If everything is set correctly, you should see a white background with a dark fringe in down the middle.  This is an interference fringe, derived from the shear between what can be thought of the real image and the reference image. That shear is created by the prism.  The reason that you don't see a ghost image in conventional DIC (which you WOULD see in other, related techniques, such as Jamin-Lebedeff), is that the shear is smaller than the resolving power of the objective.  Hence the name "differential" (small).

For the DIC background to be even, the fringe pretty much needs to fill most of the back focal plane, so the prism is engineered to fit the NA. 

Since your 40x dry is lower in NA than your oil objective, I would go ahead try the prism. You may have to adjust the compensator a bit further, but it is worth the try.  If one of the Zeiss specialists is "lurking," perhaps they can give you a more definitive answer.

I wrote a comprehensive "lay" article on DIC in American Lab, April 1988 that you might find helpful.  We've tried scanning it but have had problems. I am on the road through 10Sept but can try again after that, if you are interested.  There is also a detailed description in "Optimizing Light Microscopy," and I suspect that there will be dynamic Java tutorials on both the Olympus micro site and the Nikon uUniversity. 

By the way, because of all the mis-interpretation that comes from features in the field that respond to polarized light, I modified my instructions for DIC set up some years ago. The "Foster Method" :

1. Establish Koehler and observe any native color

2. Cross polars and observe any features that are bright against the dark background.  They will respond to the polarizing part of the system rather than the DIC beam shearing part.

3. Insert the DIC prisms (usually in the nosepiece and the MATCHING prism in the condenser)

4. Tune the compensator to soft dove gray background (the article explains why this is the most sensitive position).

Hope this is helpful.  Good hunting!

Barbara Foster, President

We've moved!

Microscopy/Microscopy Education

7101 Royal Glen Trail, Suite A

McKinney TX 75070

P: [phone]

Skype: fostermme

W: www.MicroscopyEducation.com

MME is now scheduling customized, on-site courses through December.  Call us today for details.

P. S.

Need a good general reference or light microscopy text for next semester? Call us today to learn more about "Optimizing LIght Microscopy".  Copies still available through MME... even for class-room lots ... and we give quantity discounts. Just call us here in the MME office for details.

At 09:08 PM 8/26/2007, you wrote:

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

What does the shear required by DIC depend on? Magnification or NA? To me it appears that only magnification should matter as DIC measures the phase gradient in direction of shear between two points in space. Any good reference that quantitatively relates magnification/NA to the shear required?

I wish to do time-lapse of live cells in DIC. I have a 40x 0.75 NA and 40x 1.3 NA oil objectives from Zeiss. For the 0.75 NA objective we do not have DIC prism but for 1.3NA we have (as per Zeiss nomencleture DIC III). During time-lapse I see lot of focal drift with oil objective but with air objective focus is relatively stable. Is it alright to use 1.3 NA objective's prism with 0.75 NA objective?

I have taken some images with such configuration and I think the match is not perfect - because when changing the bias retardation by moving prism I do not see transition from positive gradient to zero contrast to negative gradient.

Thanks for all inputs

Best regards

Shalin

--

My co-ordinates:

Shalin Mehta, Graduate student

Graduate Programme in Bioengineering, NUS, Singapore

Email: shalin {dot} mehta {at} gmail {dot} com

Blog: electricsbm.blogspot.com

Mobile: [phone]

--
My co-ordinates:
Shalin Mehta, Graduate student
Graduate Programme in Bioengineering, NUS, Singapore
Email: shalin {dot} mehta {at} gmail {dot} com
Blog: electricsbm.blogspot.com
Mobile: [phone]