Re: Proper Nyquist sampling with a 1.45 NA objective and a 16x16 um pixel EMCCD camera

[David Barnes-2]

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

I plugged your values into John Ossi's very neat excel "pixel calculator" and assuming you are using a 1X coupler, which I didnt see mentioned in any of the responses, you would need to magnify your image 2.65X to even reach the 2.3 sampling rate.

So you would probably need a mag changer and a camera coupler greater than 1X.

 

dave

 

On 8/1/07, John Oreopoulos <[hidden email]> wrote:

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

Someone had mentioned that the tube lens is the optovar for Olympus. I suppose instead of inserting additional lenses, I could remove the tube lens from the bottom of the trinocular, and then replace this single lens with another high quality lens, bearing in mind that the camera position then needs to change vertically along the optical axis so that everything is parfocal again.

 

Alexa had suggested buying a box of lenses and trying each one out individually until the magnification is right. Is it possible to calculate what focal length to use, where to place it and where the camera should be placed to be parfocal? Or is trial and error a simpler method? Does anyone know of a book or journal article that talks about modifying a microscope in this manner?

 

My microscope does have a 1.5x slider, and this does help a bit, but then the back projected pixel length is still only 178 nm instead of 91 nm.

 

John

 

On 1-Aug-07, at 10:10 PM, Farid Jalali wrote:

Search the CONFOCAL archive at <a onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi John,
I spoke to George from Olympus about this very issue this week, and apparently there is or there will soon be a coupling tube (I think that's what its called) that we can mount in front of the camera to give us the extra Mag needed to get us closer to Nyquist in X-Y using the 16umx16um Cascade's. You should ask him about this specifically for your IX70 frame. We have the IX81 frame and will not be able to use the part with the DSU.

Hope all is well.
Cheers
Farid

On 8/1/07, Armstrong, Brian <[hidden email]> wrote:

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

Some Olympus IX70 scopes came with a mag changer that is 1.6X, and there
is optional 2X mag changer. If your scope body does not already have the
slot for the mag changer (it is a slider) you might be able to have your
engineering dept make you one. Just a thought!
Cheers,

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
1450 E Duarte Rd
Duarte, CA 91010
626-359-8111 x62872
<a onclick="return top.js.OpenExtLink(window,event,this)" href="http://www.cityofhope.org/SharedResources/LightMicroscopy" target="_blank">http://www.cityofhope.org/SharedResources/LightMicroscopy


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of John Oreopoulos
Sent: Wednesday, August 01, 2007 10:56 AM
To: [hidden email]
Subject: Re: Proper Nyquist sampling with a 1.45 NA objective and a
16x16 um pixel EMCCD camera

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

Yes, this would be a better option, but unfortunately we can't afford
the 150x objective right now. So I think trying to retrofit a proper
expanding lens would be the next best solution.

John

On 1-Aug-07, at 12:42 PM, Julian Borejdo wrote:

> Search the CONFOCAL archive at
> <a onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank"> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> One way is to increase the magnification of the objective. Olympus now
> sells 150x/1.45 TIRF objective that uses standard immersion oil and
> coverslips.
>
> Julian Borejdo
>
>>>> John Oreopoulos <[hidden email]> 08/01/07 11:06 AM
>>>>
> Search the CONFOCAL archive at
> <a onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank"> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear list server,
>
> This topic has come up before, but I need a little more details on
> the subject. I have a 1.45 NA 60x Olympus objective mounted on an
> IX70 inverted microscope to which a Roper Cascade 512B EMCCD camera
> is attached on the trinocular. The camera has 16x16 um pixels.
> According to the Rayleigh criterion, my maximum possible resolution
> is (assuming 500 nm light):
>
> R = 0.61*lambda/NA = 0.61*500/1.45 = 210 nm
>
> According to the Nyquist sampling theorem, to properly reconstruct
> the image, I should sample the image every 210/2.3 = 91 nm in the x
> and y direction.
>
> With the 512B EMCCD camera, the back projected size of the pixel
> length using the 60x 1.45 NA objective will be:
>
> Back projected size of pixel length = real pixel length/objective
> magnification = 16/60 = 0.267 um = 267 nm
>
> Therefore, I am currently under-sampling the images. I need my back
> projected pixel length to be an additional 267 / 91 = 3x smaller.
>
> If I am wrong with any of my calculations at this point, please
> correct me. If I'm not wrong, then the only way to correct this
> problem is to magnify the image further so that features in the image
>
> can be more finely sampled by the 16x16 um pixels of the camera. For
> my application, I am not "light starved", and so I think I can afford
>
> the signal loss that will accompany the additional magnification as a
>
> result of spreading the light in the image over a larger area.
> Resolution is more critical and hence the need for better sampling.
>
> Here is my question: What kind of lens (focal length, diameter,
> apochromat, etc.) should I use to magnify the image further for
> proper Nyquist sampling, and how and where should I fit this to my
> microscope? Unfortunately my knowledge of geometric optics is very
> basic and I don't want to ask my boss to buy the wrong lens.
> Because the objective is infinity corrected, I imagine I can insert
> the lens anywhere in infinity space. The logical position is between
> the camera mount and the microscope trinocular, correct? But if I do
> that, I may have to change the vertical position of the camera so
> that the image on the camera is parfocal with the eypieces. Is there
> a suitable lens that can be used without having the extend the
> distance of the camera from the scope too much like seen in this
> youtube video:
>
> <a onclick="return top.js.OpenExtLink(window,event,this)" href="http://www.youtube.com/watch?v=9ncrx6NkXmQ&amp;mode=related&amp;search=" target="_blank"> http://www.youtube.com/watch?v=9ncrx6NkXmQ&mode=related&search=
>
> Is there a standard modular part from Olympus that can be inserted
> into the microscope that can do the magnification easily (the
> "optovar")? Would any special microscope and camera adaptors be
> needed as well? Note that the microscope that I'm using has dual use
> as a epifluorescence and TIRF microscope which both use the same
> objective. All previous posts on this topic on the list server were
> talking about spinning disk confocals I think.
>
> Once again, thanks to everyone on the list server for being such a
> great resource for microscope information. I don't know how I'd
> complete my PhD without you guys.
>
>
> 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
>
>
>
>
> ** Confidentiality Notice: This e-mail and any files transmitted
> with it are confidential to the extent permitted by law and
> intended solely for the use of the individual or entity to whom
> they are addressed. If you have received this e-mail in error
> please notify the originator of the message and destroy all copies. **

"EMF <COH.org>" made the following annotations.
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SECURITY/CONFIDENTIALITY WARNING:  This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law ( e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.
==============================================================================

--
Farid Jalali MSc
Senior Research Technician/ Lab Manager
Dr. Robert Bristow Lab
Applied Molecular Oncology
Princess Margaret Hospital
Toronto, Canada

 

[Boswell, Carl A - (cboswell)]

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

Hi Dave,

Could you provide a link or reference for "John Ossi's very neat excel "pixel calculator""?  It sounds interesting and I haven't head of it.

Thanks very much,

carl

 

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

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

From: [hidden email]

To: [hidden email]

Sent: Thursday, August 09, 2007 8:29 AM

Subject: Re: Proper Nyquist sampling with a 1.45 NA objective and a 16x16 um pixel EMCCD camera

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

I plugged your values into John Ossi's very neat excel "pixel calculator" and assuming you are using a 1X coupler, which I didnt see mentioned in any of the responses, you would need to magnify your image 2.65X to even reach the 2.3 sampling rate.

So you would probably need a mag changer and a camera coupler greater than 1X.

 

dave

 

On 8/1/07, John Oreopoulos <[hidden email]> wrote:

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

Someone had mentioned that the tube lens is the optovar for Olympus. I suppose instead of inserting additional lenses, I could remove the tube lens from the bottom of the trinocular, and then replace this single lens with another high quality lens, bearing in mind that the camera position then needs to change vertically along the optical axis so that everything is parfocal again.

 

Alexa had suggested buying a box of lenses and trying each one out individually until the magnification is right. Is it possible to calculate what focal length to use, where to place it and where the camera should be placed to be parfocal? Or is trial and error a simpler method? Does anyone know of a book or journal article that talks about modifying a microscope in this manner?

 

My microscope does have a 1.5x slider, and this does help a bit, but then the back projected pixel length is still only 178 nm instead of 91 nm.

 

John

 

On 1-Aug-07, at 10:10 PM, Farid Jalali wrote:

Search the CONFOCAL archive at <A onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target=_blank>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi John,
I spoke to George from Olympus about this very issue this week, and apparently there is or there will soon be a coupling tube (I think that's what its called) that we can mount in front of the camera to give us the extra Mag needed to get us closer to Nyquist in X-Y using the 16umx16um Cascade's. You should ask him about this specifically for your IX70 frame. We have the IX81 frame and will not be able to use the part with the DSU.

Hope all is well.
Cheers
Farid

On 8/1/07, Armstrong, Brian <[hidden email]> wrote:

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

Some Olympus IX70 scopes came with a mag changer that is 1.6X, and there
is optional 2X mag changer. If your scope body does not already have the
slot for the mag changer (it is a slider) you might be able to have your
engineering dept make you one. Just a thought!
Cheers,

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
1450 E Duarte Rd
Duarte, CA 91010
626-359-8111 x62872
<A onclick="return top.js.OpenExtLink(window,event,this)" href="http://www.cityofhope.org/SharedResources/LightMicroscopy" target=_blank>http://www.cityofhope.org/SharedResources/LightMicroscopy


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of John Oreopoulos
Sent: Wednesday, August 01, 2007 10:56 AM
To: [hidden email]
Subject: Re: Proper Nyquist sampling with a 1.45 NA objective and a
16x16 um pixel EMCCD camera

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

Yes, this would be a better option, but unfortunately we can't afford
the 150x objective right now. So I think trying to retrofit a proper
expanding lens would be the next best solution.

John

On 1-Aug-07, at 12:42 PM, Julian Borejdo wrote:

> Search the CONFOCAL archive at
> <A onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target=_blank>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> One way is to increase the magnification of the objective. Olympus now
> sells 150x/1.45 TIRF objective that uses standard immersion oil and
> coverslips.
>
> Julian Borejdo
>
>>>> John Oreopoulos <[hidden email]> 08/01/07 11:06 AM
>>>>
> Search the CONFOCAL archive at
> <A onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target=_blank>http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear list server,
>
> This topic has come up before, but I need a little more details on
> the subject. I have a 1.45 NA 60x Olympus objective mounted on an
> IX70 inverted microscope to which a Roper Cascade 512B EMCCD camera
> is attached on the trinocular. The camera has 16x16 um pixels.
> According to the Rayleigh criterion, my maximum possible resolution
> is (assuming 500 nm light):
>
> R = 0.61*lambda/NA = 0.61*500/1.45 = 210 nm
>
> According to the Nyquist sampling theorem, to properly reconstruct
> the image, I should sample the image every 210/2.3 = 91 nm in the x
> and y direction.
>
> With the 512B EMCCD camera, the back projected size of the pixel
> length using the 60x 1.45 NA objective will be:
>
> Back projected size of pixel length = real pixel length/objective
> magnification = 16/60 = 0.267 um = 267 nm
>
> Therefore, I am currently under-sampling the images. I need my back
> projected pixel length to be an additional 267 / 91 = 3x smaller.
>
> If I am wrong with any of my calculations at this point, please
> correct me. If I'm not wrong, then the only way to correct this
> problem is to magnify the image further so that features in the image
>
> can be more finely sampled by the 16x16 um pixels of the camera. For
> my application, I am not "light starved", and so I think I can afford
>
> the signal loss that will accompany the additional magnification as a
>
> result of spreading the light in the image over a larger area.
> Resolution is more critical and hence the need for better sampling.
>
> Here is my question: What kind of lens (focal length, diameter,
> apochromat, etc.) should I use to magnify the image further for
> proper Nyquist sampling, and how and where should I fit this to my
> microscope? Unfortunately my knowledge of geometric optics is very
> basic and I don't want to ask my boss to buy the wrong lens.
> Because the objective is infinity corrected, I imagine I can insert
> the lens anywhere in infinity space. The logical position is between
> the camera mount and the microscope trinocular, correct? But if I do
> that, I may have to change the vertical position of the camera so
> that the image on the camera is parfocal with the eypieces. Is there
> a suitable lens that can be used without having the extend the
> distance of the camera from the scope too much like seen in this
> youtube video:
>
> <A onclick="return top.js.OpenExtLink(window,event,this)" href="http://www.youtube.com/watch?v=9ncrx6NkXmQ&amp;mode=related&amp;search=" target=_blank>http://www.youtube.com/watch?v=9ncrx6NkXmQ&mode=related&search=
>
> Is there a standard modular part from Olympus that can be inserted
> into the microscope that can do the magnification easily (the
> "optovar")? Would any special microscope and camera adaptors be
> needed as well? Note that the microscope that I'm using has dual use
> as a epifluorescence and TIRF microscope which both use the same
> objective. All previous posts on this topic on the list server were
> talking about spinning disk confocals I think.
>
> Once again, thanks to everyone on the list server for being such a
> great resource for microscope information. I don't know how I'd
> complete my PhD without you guys.
>
>
> 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
>
>
>
>
> ** Confidentiality Notice: This e-mail and any files transmitted
> with it are confidential to the extent permitted by law and
> intended solely for the use of the individual or entity to whom
> they are addressed. If you have received this e-mail in error
> please notify the originator of the message and destroy all copies. **

"EMF <COH.org>" made the following annotations.
------------------------------------------------------------------------------

SECURITY/CONFIDENTIALITY WARNING:  This message and any attachments are intended solely for the individual or entity to which they are addressed. This communication may contain information that is privileged, confidential, or exempt from disclosure under applicable law ( e.g., personal health information, research data, financial information). Because this e-mail has been sent without encryption, individuals other than the intended recipient may be able to view the information, forward it to others or tamper with the information without the knowledge or consent of the sender. If you are not the intended recipient, or the employee or person responsible for delivering the message to the intended recipient, any dissemination, distribution or copying of the communication is strictly prohibited. If you received the communication in error, please notify the sender immediately by replying to this message and deleting the message and any accompanying files from your system. If, due to the security risks, you do not wish to receive further communications via e-mail, please reply to this message and inform the sender that you do not wish to receive further e-mail from the sender.
==============================================================================

--
Farid Jalali MSc
Senior Research Technician/ Lab Manager
Dr. Robert Bristow Lab
Applied Molecular Oncology
Princess Margaret Hospital
Toronto, Canada

 

[David Barnes-2]

Search the CONFOCAL archive at http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal you can email him at [hidden email]

On 8/9/07, Carl Boswell <[hidden email]> wrote:

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

Hi Dave,

Could you provide a link or reference for "John Ossi's very neat excel "pixel calculator""?  It sounds interesting and I haven't head of it.

Thanks very much,

carl

 

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

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

From: [hidden email]

To: [hidden email]

Sent: Thursday, August 09, 2007 8:29 AM

Subject: Re: Proper Nyquist sampling with a 1.45 NA objective and a 16x16 um pixel EMCCD camera

 

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

I plugged your values into John Ossi's very neat excel "pixel calculator" and assuming you are using a 1X coupler, which I didnt see mentioned in any of the responses, you would need to magnify your image 2.65X to even reach the 2.3 sampling rate.

So you would probably need a mag changer and a camera coupler greater than 1X.

 

dave

 

On 8/1/07, John Oreopoulos <[hidden email]> wrote:

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

Someone had mentioned that the tube lens is the optovar for Olympus. I suppose instead of inserting additional lenses, I could remove the tube lens from the bottom of the trinocular, and then replace this single lens with another high quality lens, bearing in mind that the camera position then needs to change vertically along the optical axis so that everything is parfocal again.

 

Alexa had suggested buying a box of lenses and trying each one out individually until the magnification is right. Is it possible to calculate what focal length to use, where to place it and where the camera should be placed to be parfocal? Or is trial and error a simpler method? Does anyone know of a book or journal article that talks about modifying a microscope in this manner?

 

My microscope does have a 1.5x slider, and this does help a bit, but then the back projected pixel length is still only 178 nm instead of 91 nm.

 

John

 

On 1-Aug-07, at 10:10 PM, Farid Jalali wrote:

Search the CONFOCAL archive at <a onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank">http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal Hi John,
I spoke to George from Olympus about this very issue this week, and apparently there is or there will soon be a coupling tube (I think that's what its called) that we can mount in front of the camera to give us the extra Mag needed to get us closer to Nyquist in X-Y using the 16umx16um Cascade's. You should ask him about this specifically for your IX70 frame. We have the IX81 frame and will not be able to use the part with the DSU.

Hope all is well.
Cheers
Farid

On 8/1/07, Armstrong, Brian <[hidden email]> wrote:

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

Some Olympus IX70 scopes came with a mag changer that is 1.6X, and there
is optional 2X mag changer. If your scope body does not already have the
slot for the mag changer (it is a slider) you might be able to have your
engineering dept make you one. Just a thought!
Cheers,

Brian D Armstrong PhD
Light Microscopy Core Manager
Beckman Research Institute
City of Hope
1450 E Duarte Rd
Duarte, CA 91010
626-359-8111 x62872
<a onclick="return top.js.OpenExtLink(window,event,this)" href="http://www.cityofhope.org/SharedResources/LightMicroscopy" target="_blank">http://www.cityofhope.org/SharedResources/LightMicroscopy


-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On
Behalf Of John Oreopoulos
Sent: Wednesday, August 01, 2007 10:56 AM
To: [hidden email]
Subject: Re: Proper Nyquist sampling with a 1.45 NA objective and a
16x16 um pixel EMCCD camera

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

Yes, this would be a better option, but unfortunately we can't afford
the 150x objective right now. So I think trying to retrofit a proper
expanding lens would be the next best solution.

John

On 1-Aug-07, at 12:42 PM, Julian Borejdo wrote:

> Search the CONFOCAL archive at
> <a onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank"> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> One way is to increase the magnification of the objective. Olympus now
> sells 150x/1.45 TIRF objective that uses standard immersion oil and
> coverslips.
>
> Julian Borejdo
>
>>>> John Oreopoulos <[hidden email]> 08/01/07 11:06 AM
>>>>
> Search the CONFOCAL archive at
> <a onclick="return top.js.OpenExtLink(window,event,this)" href="http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal" target="_blank"> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Dear list server,
>
> This topic has come up before, but I need a little more details on
> the subject. I have a 1.45 NA 60x Olympus objective mounted on an
> IX70 inverted microscope to which a Roper Cascade 512B EMCCD camera
> is attached on the trinocular. The camera has 16x16 um pixels.
> According to the Rayleigh criterion, my maximum possible resolution
> is (assuming 500 nm light):
>
> R = 0.61*lambda/NA = 0.61*500/1.45 = 210 nm
>
> According to the Nyquist sampling theorem, to properly reconstruct
> the image, I should sample the image every 210/2.3 = 91 nm in the x
> and y direction.
>
> With the 512B EMCCD camera, the back projected size of the pixel
> length using the 60x 1.45 NA objective will be:
>
> Back projected size of pixel length = real pixel length/objective
> magnification = 16/60 = 0.267 um = 267 nm
>
> Therefore, I am currently under-sampling the images. I need my back
> projected pixel length to be an additional 267 / 91 = 3x smaller.
>
> If I am wrong with any of my calculations at this point, please
> correct me. If I'm not wrong, then the only way to correct this
> problem is to magnify the image further so that features in the image
>
> can be more finely sampled by the 16x16 um pixels of the camera. For
> my application, I am not "light starved", and so I think I can afford
>
> the signal loss that will accompany the additional magnification as a
>
> result of spreading the light in the image over a larger area.
> Resolution is more critical and hence the need for better sampling.
>
> Here is my question: What kind of lens (focal length, diameter,
> apochromat, etc.) should I use to magnify the image further for
> proper Nyquist sampling, and how and where should I fit this to my
> microscope? Unfortunately my knowledge of geometric optics is very
> basic and I don't want to ask my boss to buy the wrong lens.
> Because the objective is infinity corrected, I imagine I can insert
> the lens anywhere in infinity space. The logical position is between
> the camera mount and the microscope trinocular, correct? But if I do
> that, I may have to change the vertical position of the camera so
> that the image on the camera is parfocal with the eypieces. Is there
> a suitable lens that can be used without having the extend the
> distance of the camera from the scope too much like seen in this
> youtube video:
>
> <a onclick="return top.js.OpenExtLink(window,event,this)" href="http://www.youtube.com/watch?v=9ncrx6NkXmQ&amp;mode=related&amp;search=" target="_blank"> http://www.youtube.com/watch?v=9ncrx6NkXmQ&mode=related&search=
>
> Is there a standard modular part from Olympus that can be inserted
> into the microscope that can do the magnification easily (the
> "optovar")? Would any special microscope and camera adaptors be
> needed as well? Note that the microscope that I'm using has dual use
> as a epifluorescence and TIRF microscope which both use the same
> objective. All previous posts on this topic on the list server were
> talking about spinning disk confocals I think.
>
> Once again, thanks to everyone on the list server for being such a
> great resource for microscope information. I don't know how I'd
> complete my PhD without you guys.
>
>
> 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
>
>
>
>
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--
Farid Jalali MSc
Senior Research Technician/ Lab Manager
Dr. Robert Bristow Lab
Applied Molecular Oncology
Princess Margaret Hospital
Toronto, Canada