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At what confocal pinhole size does a confocal image cease to be confocal?

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John Oreopoulos

At what confocal pinhole size does a confocal image cease to be confocal?

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I'm wondering if the listserver can help me recall something I've heard several times before and point me to a reference that proves (and demonstrates) it. Several people I think have said before (I'm looking in the direction of Jim Pawley and Guy Cox perhaps) that beyond a certain confocal pinhole size (in terms of Airy units), the image that is produced by the confocal microscope is equivalent to that which would be obtained with a regular widefield epifluorescence microscope without confocal optics.

1. Can someone tell me at what confocal pinhole size this happens?
2. Can you also tell me where this was shown in the confocal literature for the first time (or a equivalently in a review article on same the topic)?
3. Does this principle also apply to spinning disk confocal microscopes, why or why not?

Thank you greatly for your wisdom.

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca

mmodel

Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

Hi John.
There is a chapter about pinhole size in the 2nd edition of the Handbook (Sandison et al, pp. 39-53), and you may also check out Wilson's papers (Confocal Microscopy, Academic Press, 1990, ed. T. Wilson; chapter 1).
Best wishes

Mike Model

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Tuesday, January 28, 2014 11:28 AM
To: [hidden email]
Subject: At what confocal pinhole size does a confocal image cease to be confocal?

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

I'm wondering if the listserver can help me recall something I've heard several times before and point me to a reference that proves (and demonstrates) it. Several people I think have said before (I'm looking in the direction of Jim Pawley and Guy Cox perhaps) that beyond a certain confocal pinhole size (in terms of Airy units), the image that is produced by the confocal microscope is equivalent to that which would be obtained with a regular widefield epifluorescence microscope without confocal optics.

1. Can someone tell me at what confocal pinhole size this happens?
2. Can you also tell me where this was shown in the confocal literature for the first time (or a equivalently in a review article on same the topic)?
3. Does this principle also apply to spinning disk confocal microscopes, why or why not?

Thank you greatly for your wisdom.

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca

MORONE Diego RIC

Re: At what confocal pinhole size does a confocal image cease to be confocal?

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*****

Regarding the question on the first paper, I think you'll need to go back to Marvin Minsky's PhD thesis in 1955. I couldn't find the text, but this is a nice read anyway

http://web.media.mit.edu/~minsky/papers/ConfocalMemoir.html

(slightly off topic: here's the patent he filed in 1957)

https://www.google.com/patents/US3013467?dq=minsky&hl=en&sa=X&ei=4N7nUuzOB4iIyAOV4IDQAQ&ved=0CDoQ6AEwAQ

Best
Diego

................................
Diego Morone
Research Center
ISTITUTO CLINICO HUMANITAS
via A. Manzoni, 113 - 20089 Rozzano - Milano, Italy
Phone: +39028224-5252, +39028224-5149 - Fax: +39028224-5290
Email: [hidden email]
Map and directions: http://goo.gl/maps/pVWbq
www.humanitas.it/hur/cms/english/

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-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of MODEL, MICHAEL
Sent: martedì 28 gennaio 2014 17:56
To: [hidden email]
Subject: Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

Hi John.
There is a chapter about pinhole size in the 2nd edition of the Handbook (Sandison et al, pp. 39-53), and you may also check out Wilson's papers (Confocal Microscopy, Academic Press, 1990, ed. T. Wilson; chapter 1).
Best wishes

Mike Model

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of John Oreopoulos
Sent: Tuesday, January 28, 2014 11:28 AM
To: [hidden email]
Subject: At what confocal pinhole size does a confocal image cease to be confocal?

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

I'm wondering if the listserver can help me recall something I've heard several times before and point me to a reference that proves (and demonstrates) it. Several people I think have said before (I'm looking in the direction of Jim Pawley and Guy Cox perhaps) that beyond a certain confocal pinhole size (in terms of Airy units), the image that is produced by the confocal microscope is equivalent to that which would be obtained with a regular widefield epifluorescence microscope without confocal optics.

1. Can someone tell me at what confocal pinhole size this happens?
2. Can you also tell me where this was shown in the confocal literature for the first time (or a equivalently in a review article on same the topic)?
3. Does this principle also apply to spinning disk confocal microscopes, why or why not?

Thank you greatly for your wisdom.

John Oreopoulos
Staff Scientist
Spectral Applied Research Inc.
A Division of Andor Technology
Richmond Hill, Ontario
Canada
www.spectral.ca

Turnbull, Lon

Re: At what confocal pinhole size does a confocal image cease to be confocal?

In reply to this post by John Oreopoulos

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

Hi John,

I do not know the answer to your first questions, but I can give you an answer to your third question.
Spinning disk systems that I am familiar with have a fixed pinhole size that is optimized for a particular objective, usually the highest power objective. Therefore, the lower power objectives have a larger optical section. With a small enough power objective, for example, the optical section becomes very large and might even be considered to be non-confocal. For example, if a 100x objective has a 0.91 micrometer optical section then on the same system a 5x objective has a 95 micrometer optical section.

Lon

Microscope manager
Department of Biological Sciences
University of North Texas

Sent from my iPad

> On Jan 28, 2014, at 11:04 AM, "John Oreopoulos" <[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I'm wondering if the listserver can help me recall something I've heard several times before and point me to a reference that proves (and demonstrates) it. Several people I think have said before (I'm looking in the direction of Jim Pawley and Guy Cox perhaps) that beyond a certain confocal pinhole size (in terms of Airy units), the image that is produced by the confocal microscope is equivalent to that which would be obtained with a regular widefield epifluorescence microscope without confocal optics.
>
> 1. Can someone tell me at what confocal pinhole size this happens?
> 2. Can you also tell me where this was shown in the confocal literature for the first time (or a equivalently in a review article on same the topic)?
> 3. Does this principle also apply to spinning disk confocal microscopes, why or why not?
>
> Thank you greatly for your wisdom.
>
>
> John Oreopoulos
> Staff Scientist
> Spectral Applied Research Inc.
> A Division of Andor Technology
> Richmond Hill, Ontario
> Canada
> www.spectral.ca

Guy Cox-2

Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

There is no one answer. A confocal microscope with a pinhole of absolutely any size will provide more optical sectioning than a wide-field microscope. The smaller the pinhole the better the optical sectioning. However once one goes much below one Airy diameter the loss in signal will pretty much negate any further gain. The question that has more relevance is where can we get the sqrt2 increase in lateral resolution that confocal potentially provides? The answer is that to get the whole value we need an infinitely small pinhole, and one the pinhole reaches one Airy value the resolution is the same as wide-field.

Guy Cox & Colin Sheppard, 2004. Practical limits of resolution in confocal and non-linear microscopy. Microscopy Research & Technique, 63, 18-22

I've really lost count of how many times I've posted this reference!

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Turnbull, Lon
Sent: Wednesday, 29 January 2014 4:39 AM
To: [hidden email]
Subject: Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

Hi John,

I do not know the answer to your first questions, but I can give you an answer to your third question.
Spinning disk systems that I am familiar with have a fixed pinhole size that is optimized for a particular objective, usually the highest power objective. Therefore, the lower power objectives have a larger optical section. With a small enough power objective, for example, the optical section becomes very large and might even be considered to be non-confocal. For example, if a 100x objective has a 0.91 micrometer optical section then on the same system a 5x objective has a 95 micrometer optical section.

Lon

Microscope manager
Department of Biological Sciences
University of North Texas

Sent from my iPad

Armstrong, Brian

Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

Hi John, for the theoretical limits of the gain in resolution you get from using Confocal you could see Pawley's book ref mentioned previously. For a practical limit; the gain from the Confocal is from the creation of the optical slice through the use of a pinhole that excludes out of focus light and therefore may increase the "resolution" of the image produced (more signal / less noise). However, this depends upon the sample. If there is no out of focus light beyond the range of the optical slice then there is no gain in the Confocal image. Moreover, if your section is cut physically thinner than the optical slice the widefield image and Confocal image should be identical with 1 Airy unit pinhole size. In other words, it wouldn't make much sense to use a Confocal microscope to image a 1um thick tissue section (in most cases).
So, in my opinion, the answer is that it is largely sample dependent.
*I imagine that you know this already.
Cheers,

Brian D Armstrong PhD
Associate Research Professor
Director, Light Microscopy Core
Beckman Research Institute
City of Hope
Dept of Neuroscience
1450 E Duarte Rd
Duarte, CA 91010
626-256-4673 x62872

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
Sent: Wednesday, January 29, 2014 5:59 AM
To: [hidden email]
Subject: Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

There is no one answer. A confocal microscope with a pinhole of absolutely any size will provide more optical sectioning than a wide-field microscope. The smaller the pinhole the better the optical sectioning. However once one goes much below one Airy diameter the loss in signal will pretty much negate any further gain. The question that has more relevance is where can we get the sqrt2 increase in lateral resolution that confocal potentially provides? The answer is that to get the whole value we need an infinitely small pinhole, and one the pinhole reaches one Airy value the resolution is the same as wide-field.

Guy Cox & Colin Sheppard, 2004. Practical limits of resolution in confocal and non-linear microscopy. Microscopy Research & Technique, 63, 18-22

I've really lost count of how many times I've posted this reference!

Guy

-----Original Message-----
From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Turnbull, Lon
Sent: Wednesday, 29 January 2014 4:39 AM
To: [hidden email]
Subject: Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

Hi John,

I do not know the answer to your first questions, but I can give you an answer to your third question.
Spinning disk systems that I am familiar with have a fixed pinhole size that is optimized for a particular objective, usually the highest power objective. Therefore, the lower power objectives have a larger optical section. With a small enough power objective, for example, the optical section becomes very large and might even be considered to be non-confocal. For example, if a 100x objective has a 0.91 micrometer optical section then on the same system a 5x objective has a 95 micrometer optical section.

Lon

Microscope manager
Department of Biological Sciences
University of North Texas

Sent from my iPad

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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. (fpc5p)
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John Oreopoulos

Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

Hi Brian, yes I guess this gets back to the discussion I started a few months back that led to an explanation about the differences between "resolution" and "optical sectioning". My question to the listserver was motivated by the observation that more often than not, one would like to keep the specimen alive and use a little laser power as possible to image the sample. A good way to achieve this is to turn down the laser power and open up the confocal pinhole, thereby sacrificing some degree optical sectioning. What surprises me is just how much one can actually open up the pinhole sometimes and still get a "confocal enough" image that shows the features of interest with good detail, and it is still far superior compared to the widefield image. As you say, this is probably somewhat dependent on the structure of interest in the cell/tissue that's being imaged.

Does anyone know if there is a way to express the loss of optical sectioning ability as a function of pinhole size above 1 Airy unit? Can this be quantified? Is it a linear trend?

John

On 2014-01-29, at 11:59 AM, Armstrong, Brian wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi John, for the theoretical limits of the gain in resolution you get from using Confocal you could see Pawley's book ref mentioned previously. For a practical limit; the gain from the Confocal is from the creation of the optical slice through the use of a pinhole that excludes out of focus light and therefore may increase the "resolution" of the image produced (more signal / less noise). However, this depends upon the sample. If there is no out of focus light beyond the range of the optical slice then there is no gain in the Confocal image. Moreover, if your section is cut physically thinner than the optical slice the widefield image and Confocal image should be identical with 1 Airy unit pinhole size. In other words, it wouldn't make much sense to use a Confocal microscope to image a 1um thick tissue section (in most cases).
> So, in my opinion, the answer is that it is largely sample dependent.
> *I imagine that you know this already.
> Cheers,
>
> Brian D Armstrong PhD
> Associate Research Professor
> Director, Light Microscopy Core
> Beckman Research Institute
> City of Hope
> Dept of Neuroscience
> 1450 E Duarte Rd
> Duarte, CA 91010
> 626-256-4673 x62872
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
> Sent: Wednesday, January 29, 2014 5:59 AM
> To: [hidden email]
> Subject: Re: At what confocal pinhole size does a confocal image cease to be confocal?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> There is no one answer. A confocal microscope with a pinhole of absolutely any size will provide more optical sectioning than a wide-field microscope. The smaller the pinhole the better the optical sectioning. However once one goes much below one Airy diameter the loss in signal will pretty much negate any further gain. The question that has more relevance is where can we get the sqrt2 increase in lateral resolution that confocal potentially provides? The answer is that to get the whole value we need an infinitely small pinhole, and one the pinhole reaches one Airy value the resolution is the same as wide-field.
>
> Guy Cox & Colin Sheppard, 2004. Practical limits of resolution in confocal and non-linear microscopy. Microscopy Research & Technique, 63, 18-22
>
> I've really lost count of how many times I've posted this reference!
>
> Guy
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Turnbull, Lon
> Sent: Wednesday, 29 January 2014 4:39 AM
> To: [hidden email]
> Subject: Re: At what confocal pinhole size does a confocal image cease to be confocal?
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi John,
>
> I do not know the answer to your first questions, but I can give you an answer to your third question.
> Spinning disk systems that I am familiar with have a fixed pinhole size that is optimized for a particular objective, usually the highest power objective. Therefore, the lower power objectives have a larger optical section. With a small enough power objective, for example, the optical section becomes very large and might even be considered to be non-confocal. For example, if a 100x objective has a 0.91 micrometer optical section then on the same system a 5x objective has a 95 micrometer optical section.
>
>
> Lon
>
> Microscope manager
> Department of Biological Sciences
> University of North Texas
>
>
> Sent from my iPad
>
>> On Jan 28, 2014, at 11:04 AM, "John Oreopoulos" <[hidden email]> wrote:
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> I'm wondering if the listserver can help me recall something I've heard several times before and point me to a reference that proves (and demonstrates) it. Several people I think have said before (I'm looking in the direction of Jim Pawley and Guy Cox perhaps) that beyond a certain confocal pinhole size (in terms of Airy units), the image that is produced by the confocal microscope is equivalent to that which would be obtained with a regular widefield epifluorescence microscope without confocal optics.
>>
>> 1. Can someone tell me at what confocal pinhole size this happens?
>> 2. Can you also tell me where this was shown in the confocal literature for the first time (or a equivalently in a review article on same the topic)?
>> 3. Does this principle also apply to spinning disk confocal microscopes, why or why not?
>>
>> Thank you greatly for your wisdom.
>>
>>
>> John Oreopoulos
>> Staff Scientist
>> Spectral Applied Research Inc.
>> A Division of Andor Technology
>> Richmond Hill, Ontario
>> Canada
>> www.spectral.ca
>
>
> ---------------------------------------------------------------------
> *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. (fpc5p)
> ---------------------------------------------------------------------

Julio Vazquez

Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

Hi John,

In addition to all references already provided, there is also a good discussion of these issues (resolution, confocality) in the Zeiss publication: Confocal Laser Scanning Microscopy Principles; the paper can be downloaded from the Zeiss Campus website (if link below is not good, just Google the title)

http://zeiss-campus.magnet.fsu.edu/referencelibrary/pdfs/ZeissConfocalPrinciples.pdf

Julio.
--
Julio Vazquez
Fred Hutchinson Cancer Research Center
Seattle, WA 98109

http://www.fhcrc.org/en.html

On Jan 29, 2014, at 9:19 AM, John Oreopoulos wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Brian, yes I guess this gets back to the discussion I started a few months back that led to an explanation about the differences between "resolution" and "optical sectioning". My question to the listserver was motivated by the observation that more often than not, one would like to keep the specimen alive and use a little laser power as possible to image the sample. A good way to achieve this is to turn down the laser power and open up the confocal pinhole, thereby sacrificing some degree optical sectioning. What surprises me is just how much one can actually open up the pinhole sometimes and still get a "confocal enough" image that shows the features of interest with good detail, and it is still far superior compared to the widefield image. As you say, this is probably somewhat dependent on the structure of interest in the cell/tissue that's being imaged.
>
> Does anyone know if there is a way to express the loss of optical sectioning ability as a function of pinhole size above 1 Airy unit? Can this be quantified? Is it a linear trend?
>
> John
>
>
> On 2014-01-29, at 11:59 AM, Armstrong, Brian wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi John, for the theoretical limits of the gain in resolution you get from using Confocal you could see Pawley's book ref mentioned previously. For a practical limit; the gain from the Confocal is from the creation of the optical slice through the use of a pinhole that excludes out of focus light and therefore may increase the "resolution" of the image produced (more signal / less noise). However, this depends upon the sample. If there is no out of focus light beyond the range of the optical slice then there is no gain in the Confocal image. Moreover, if your section is cut physically thinner than the optical slice the widefield image and Confocal image should be identical with 1 Airy unit pinhole size. In other words, it wouldn't make much sense to use a Confocal microscope to image a 1um thick tissue section (in most cases).
>> So, in my opinion, the answer is that it is largely sample dependent.
>> *I imagine that you know this already.
>> Cheers,
>>
>> Brian D Armstrong PhD
>> Associate Research Professor
>> Director, Light Microscopy Core
>> Beckman Research Institute
>> City of Hope
>> Dept of Neuroscience
>> 1450 E Duarte Rd
>> Duarte, CA 91010
>> 626-256-4673 x62872
>>
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
>> Sent: Wednesday, January 29, 2014 5:59 AM
>> To: [hidden email]
>> Subject: Re: At what confocal pinhole size does a confocal image cease to be confocal?
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> There is no one answer. A confocal microscope with a pinhole of absolutely any size will provide more optical sectioning than a wide-field microscope. The smaller the pinhole the better the optical sectioning. However once one goes much below one Airy diameter the loss in signal will pretty much negate any further gain. The question that has more relevance is where can we get the sqrt2 increase in lateral resolution that confocal potentially provides? The answer is that to get the whole value we need an infinitely small pinhole, and one the pinhole reaches one Airy value the resolution is the same as wide-field.
>>
>> Guy Cox & Colin Sheppard, 2004. Practical limits of resolution in confocal and non-linear microscopy. Microscopy Research & Technique, 63, 18-22
>>
>> I've really lost count of how many times I've posted this reference!
>>
>> Guy
>>
>> -----Original Message-----
>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Turnbull, Lon
>> Sent: Wednesday, 29 January 2014 4:39 AM
>> To: [hidden email]
>> Subject: Re: At what confocal pinhole size does a confocal image cease to be confocal?
>>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi John,
>>
>> I do not know the answer to your first questions, but I can give you an answer to your third question.
>> Spinning disk systems that I am familiar with have a fixed pinhole size that is optimized for a particular objective, usually the highest power objective. Therefore, the lower power objectives have a larger optical section. With a small enough power objective, for example, the optical section becomes very large and might even be considered to be non-confocal. For example, if a 100x objective has a 0.91 micrometer optical section then on the same system a 5x objective has a 95 micrometer optical section.
>>
>>
>> Lon
>>
>> Microscope manager
>> Department of Biological Sciences
>> University of North Texas
>>
>>
>> Sent from my iPad
>>
>>> On Jan 28, 2014, at 11:04 AM, "John Oreopoulos" <[hidden email]> wrote:
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> I'm wondering if the listserver can help me recall something I've heard several times before and point me to a reference that proves (and demonstrates) it. Several people I think have said before (I'm looking in the direction of Jim Pawley and Guy Cox perhaps) that beyond a certain confocal pinhole size (in terms of Airy units), the image that is produced by the confocal microscope is equivalent to that which would be obtained with a regular widefield epifluorescence microscope without confocal optics.
>>>
>>> 1. Can someone tell me at what confocal pinhole size this happens?
>>> 2. Can you also tell me where this was shown in the confocal literature for the first time (or a equivalently in a review article on same the topic)?
>>> 3. Does this principle also apply to spinning disk confocal microscopes, why or why not?
>>>
>>> Thank you greatly for your wisdom.
>>>
>>>
>>> John Oreopoulos
>>> Staff Scientist
>>> Spectral Applied Research Inc.
>>> A Division of Andor Technology
>>> Richmond Hill, Ontario
>>> Canada
>>> www.spectral.ca
>>
>>
>> ---------------------------------------------------------------------
>> *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. (fpc5p)
>> ---------------------------------------------------------------------

John Oreopoulos

Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

Thanks Julio, I think the equation I'm looking for is in this document. Does anyone know if equation (4) in this document is derived anywhere in the literature? Is it in any of the books that people have mentioned previously? I'd like to get a copy of that for my records.

John

On 2014-01-29, at 12:35 PM, Julio Vazquez wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi John,
>
> In addition to all references already provided, there is also a good discussion of these issues (resolution, confocality) in the Zeiss publication: Confocal Laser Scanning Microscopy Principles; the paper can be downloaded from the Zeiss Campus website (if link below is not good, just Google the title)
>
> http://zeiss-campus.magnet.fsu.edu/referencelibrary/pdfs/ZeissConfocalPrinciples.pdf
>
> Julio.
> --
> Julio Vazquez
> Fred Hutchinson Cancer Research Center
> Seattle, WA 98109
>
> http://www.fhcrc.org/en.html
>
>
>
>
>
> On Jan 29, 2014, at 9:19 AM, John Oreopoulos wrote:
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Brian, yes I guess this gets back to the discussion I started a few months back that led to an explanation about the differences between "resolution" and "optical sectioning". My question to the listserver was motivated by the observation that more often than not, one would like to keep the specimen alive and use a little laser power as possible to image the sample. A good way to achieve this is to turn down the laser power and open up the confocal pinhole, thereby sacrificing some degree optical sectioning. What surprises me is just how much one can actually open up the pinhole sometimes and still get a "confocal enough" image that shows the features of interest with good detail, and it is still far superior compared to the widefield image. As you say, this is probably somewhat dependent on the structure of interest in the cell/tissue that's being imaged.
>>
>> Does anyone know if there is a way to express the loss of optical sectioning ability as a function of pinhole size above 1 Airy unit? Can this be quantified? Is it a linear trend?
>>
>> John
>>
>>
>> On 2014-01-29, at 11:59 AM, Armstrong, Brian wrote:
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi John, for the theoretical limits of the gain in resolution you get from using Confocal you could see Pawley's book ref mentioned previously. For a practical limit; the gain from the Confocal is from the creation of the optical slice through the use of a pinhole that excludes out of focus light and therefore may increase the "resolution" of the image produced (more signal / less noise). However, this depends upon the sample. If there is no out of focus light beyond the range of the optical slice then there is no gain in the Confocal image. Moreover, if your section is cut physically thinner than the optical slice the widefield image and Confocal image should be identical with 1 Airy unit pinhole size. In other words, it wouldn't make much sense to use a Confocal microscope to image a 1um thick tissue section (in most cases).
>>> So, in my opinion, the answer is that it is largely sample dependent.
>>> *I imagine that you know this already.
>>> Cheers,
>>>
>>> Brian D Armstrong PhD
>>> Associate Research Professor
>>> Director, Light Microscopy Core
>>> Beckman Research Institute
>>> City of Hope
>>> Dept of Neuroscience
>>> 1450 E Duarte Rd
>>> Duarte, CA 91010
>>> 626-256-4673 x62872
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Guy Cox
>>> Sent: Wednesday, January 29, 2014 5:59 AM
>>> To: [hidden email]
>>> Subject: Re: At what confocal pinhole size does a confocal image cease to be confocal?
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> There is no one answer. A confocal microscope with a pinhole of absolutely any size will provide more optical sectioning than a wide-field microscope. The smaller the pinhole the better the optical sectioning. However once one goes much below one Airy diameter the loss in signal will pretty much negate any further gain. The question that has more relevance is where can we get the sqrt2 increase in lateral resolution that confocal potentially provides? The answer is that to get the whole value we need an infinitely small pinhole, and one the pinhole reaches one Airy value the resolution is the same as wide-field.
>>>
>>> Guy Cox & Colin Sheppard, 2004. Practical limits of resolution in confocal and non-linear microscopy. Microscopy Research & Technique, 63, 18-22
>>>
>>> I've really lost count of how many times I've posted this reference!
>>>
>>> Guy
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]] On Behalf Of Turnbull, Lon
>>> Sent: Wednesday, 29 January 2014 4:39 AM
>>> To: [hidden email]
>>> Subject: Re: At what confocal pinhole size does a confocal image cease to be confocal?
>>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi John,
>>>
>>> I do not know the answer to your first questions, but I can give you an answer to your third question.
>>> Spinning disk systems that I am familiar with have a fixed pinhole size that is optimized for a particular objective, usually the highest power objective. Therefore, the lower power objectives have a larger optical section. With a small enough power objective, for example, the optical section becomes very large and might even be considered to be non-confocal. For example, if a 100x objective has a 0.91 micrometer optical section then on the same system a 5x objective has a 95 micrometer optical section.
>>>
>>>
>>> Lon
>>>
>>> Microscope manager
>>> Department of Biological Sciences
>>> University of North Texas
>>>
>>>
>>> Sent from my iPad
>>>
>>>> On Jan 28, 2014, at 11:04 AM, "John Oreopoulos" <[hidden email]> wrote:
>>>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> I'm wondering if the listserver can help me recall something I've heard several times before and point me to a reference that proves (and demonstrates) it. Several people I think have said before (I'm looking in the direction of Jim Pawley and Guy Cox perhaps) that beyond a certain confocal pinhole size (in terms of Airy units), the image that is produced by the confocal microscope is equivalent to that which would be obtained with a regular widefield epifluorescence microscope without confocal optics.
>>>>
>>>> 1. Can someone tell me at what confocal pinhole size this happens?
>>>> 2. Can you also tell me where this was shown in the confocal literature for the first time (or a equivalently in a review article on same the topic)?
>>>> 3. Does this principle also apply to spinning disk confocal microscopes, why or why not?
>>>>
>>>> Thank you greatly for your wisdom.
>>>>
>>>>
>>>> John Oreopoulos
>>>> Staff Scientist
>>>> Spectral Applied Research Inc.
>>>> A Division of Andor Technology
>>>> Richmond Hill, Ontario
>>>> Canada
>>>> www.spectral.ca
>>>
>>>
>>> ---------------------------------------------------------------------
>>> *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. (fpc5p)
>>> ---------------------------------------------------------------------

Julio Vazquez

Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

Yu may want to have a look at these three:

Robert H. Webb: Confocal optical microscopy. Rep Prog Phys 59: 427-471 (1996). (can be Googled; can send you a copy if you like)

Appl Opt. 1994 Feb 1;33(4):603-15. doi: 10.1364/AO.33.000603.
Background rejection and signal-to-noise optimization in confocal and alternative fluorescence microscopes.
Sandison DR, Webb WW.
Abstract
In the confocal microscope, tightly focused illumination and spatially filtered detection are combined to reduce out-of-focus background and to produce high-quality images that display thin optical sections within thick fluorescent specimens. We define background as the detected light that originates outside a resolution volume and signal as the detected light that originates within the same volume. Background rejection is measured by the signal-to-background ratio (S/B) and is calculated for confocal, spinning-disk, line-illumination, slit-detection, and conventional fluorescence microscopes as a function of both the spatial filter size and the specimen thickness. Spatial filter sizes that reject background and optimize the signal-to-noise ratio (S/N) are calculated for each microscope. These calculations are normalized so that the time-averaged illumination at each point in the specimen is the same for each microscope. For thick specimens, we show that the S/B obtained with a confocal microscope can be more than 100 times greater than the S/B available with a conventional microscope, and we find that the optimal confocal S/N can be a factor of 10 greater than the S/N in the conventional microscope.

Appl Opt. 1995 Jul 1;34(19):3576-88. doi: 10.1364/AO.34.003576.
Quantitative comparison of background rejection, signal-to-noise ratio, and resolution in confocal and full-field laser scanning microscopes.
Sandison DR, Piston DW, Williams RM, Webb WW.
Abstract
Development of a laser scanning microscope for simultaneous three-dimensional imaging in both a full-field laser scanning mode (FLSM) and a confocal laser scanning mode (CLSM) permits the direct comparison of axial resolution and out-of-focus background rejection as a function of sample thickness for both FLSM and CLSM with varying detector aperture (pinhole) radii. The sample-dependent detector aperture radii that optimize the signal-to-noise ratio (S/N) in the CLSM are experimentally determined. The results verify earlier calculations [Appl. Opt. 33, 603 (1994)]. Using these results, we discuss the practical and theoretical limits on the S/N in the CLSM and compare them with those of a full-field epifluorescence microscope (FEM) that is enhanced by image deconvolution. The specimen volume over which the FLSM exhibits imaging properties that are equivalent to a FEM is calculated in the appendices.

Julio
--

On Jan 29, 2014, at 9:45 AM, John Oreopoulos wrote:

> Thanks Julio, I think the equation I'm looking for is in this document. Does anyone know if equation (4) in this document is derived anywhere in the literature? Is it in any of the books that people have mentioned previously? I'd like to get a copy of that for my records.
>
> John

phil laissue

Re: At what confocal pinhole size does a confocal image cease to be confocal?

In reply to this post by John Oreopoulos

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

Hi John,

it's looks bilinear, according to an earlier post by Brad Amos. See p 20
here:

http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf

On a related note, I had talked to several manufacturers a few years ago
about this new trend of using one pinhole size for different wavelengths,
and why they were not bothered about differing section thickness. I was
told by all that 'tests were performed on biological samples and it was
found that in practice the majority of samples yielded very similar results
when imaged with one versus multiple pinholes', and the alignment of
multiple pinholes was too much of a pain anyway. Having said that, some of
the major manufacturers (sadly not all) have meanwhile implemented
framescans with varying pinhole, which doesn't fully address the issue due
to the time delay. An alternative would be a fast-switching pinhole for
line scans, but I don't think anyone's looking into that.

Cheers

Philippe

On 29 January 2014 17:19, John Oreopoulos <[hidden email]>wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi Brian, yes I guess this gets back to the discussion I started a few
> months back that led to an explanation about the differences between
> "resolution" and "optical sectioning". My question to the listserver was
> motivated by the observation that more often than not, one would like to
> keep the specimen alive and use a little laser power as possible to image
> the sample. A good way to achieve this is to turn down the laser power and
> open up the confocal pinhole, thereby sacrificing some degree optical
> sectioning. What surprises me is just how much one can actually open up the
> pinhole sometimes and still get a "confocal enough" image that shows the
> features of interest with good detail, and it is still far superior
> compared to the widefield image. As you say, this is probably somewhat
> dependent on the structure of interest in the cell/tissue that's being
> imaged.
>
> Does anyone know if there is a way to express the loss of optical
> sectioning ability as a function of pinhole size above 1 Airy unit? Can
> this be quantified? Is it a linear trend?
>
> John
>
>
> On 2014-01-29, at 11:59 AM, Armstrong, Brian wrote:
>
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi John, for the theoretical limits of the gain in resolution you get
> from using Confocal you could see Pawley's book ref mentioned previously.
> For a practical limit; the gain from the Confocal is from the creation of
> the optical slice through the use of a pinhole that excludes out of focus
> light and therefore may increase the "resolution" of the image produced
> (more signal / less noise). However, this depends upon the sample. If there
> is no out of focus light beyond the range of the optical slice then there
> is no gain in the Confocal image. Moreover, if your section is cut
> physically thinner than the optical slice the widefield image and Confocal
> image should be identical with 1 Airy unit pinhole size. In other words, it
> wouldn't make much sense to use a Confocal microscope to image a 1um thick
> tissue section (in most cases).
> > So, in my opinion, the answer is that it is largely sample dependent.
> > *I imagine that you know this already.
> > Cheers,
> >
> > Brian D Armstrong PhD
> > Associate Research Professor
> > Director, Light Microscopy Core
> > Beckman Research Institute
> > City of Hope
> > Dept of Neuroscience
> > 1450 E Duarte Rd
> > Duarte, CA 91010
> > 626-256-4673 x62872
> >
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Guy Cox
> > Sent: Wednesday, January 29, 2014 5:59 AM
> > To: [hidden email]
> > Subject: Re: At what confocal pinhole size does a confocal image cease
> to be confocal?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > There is no one answer. A confocal microscope with a pinhole of
> absolutely any size will provide more optical sectioning than a wide-field
> microscope. The smaller the pinhole the better the optical sectioning.
> However once one goes much below one Airy diameter the loss in signal will
> pretty much negate any further gain. The question that has more relevance
> is where can we get the sqrt2 increase in lateral resolution that confocal
> potentially provides? The answer is that to get the whole value we need an
> infinitely small pinhole, and one the pinhole reaches one Airy value the
> resolution is the same as wide-field.
> >
> > Guy Cox & Colin Sheppard, 2004. Practical limits of resolution in
> confocal and non-linear microscopy. Microscopy Research & Technique, 63,
> 18-22
> >
> > I've really lost count of how many times I've posted this reference!
> >
> > Guy
> >
> > -----Original Message-----
> > From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of Turnbull, Lon
> > Sent: Wednesday, 29 January 2014 4:39 AM
> > To: [hidden email]
> > Subject: Re: At what confocal pinhole size does a confocal image cease
> to be confocal?
> >
> > *****
> > To join, leave or search the confocal microscopy listserv, go to:
> > http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> > *****
> >
> > Hi John,
> >
> > I do not know the answer to your first questions, but I can give you an
> answer to your third question.
> > Spinning disk systems that I am familiar with have a fixed pinhole size
> that is optimized for a particular objective, usually the highest power
> objective. Therefore, the lower power objectives have a larger optical
> section. With a small enough power objective, for example, the optical
> section becomes very large and might even be considered to be non-confocal.
> For example, if a 100x objective has a 0.91 micrometer optical section
> then on the same system a 5x objective has a 95 micrometer optical section.
> >
> >
> > Lon
> >
> > Microscope manager
> > Department of Biological Sciences
> > University of North Texas
> >
> >
> > Sent from my iPad
> >
> >> On Jan 28, 2014, at 11:04 AM, "John Oreopoulos" <
> [hidden email]> wrote:
> >>
> >> *****
> >> To join, leave or search the confocal microscopy listserv, go to:
> >> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> >> *****
> >>
> >> I'm wondering if the listserver can help me recall something I've heard
> several times before and point me to a reference that proves (and
> demonstrates) it. Several people I think have said before (I'm looking in
> the direction of Jim Pawley and Guy Cox perhaps) that beyond a certain
> confocal pinhole size (in terms of Airy units), the image that is produced
> by the confocal microscope is equivalent to that which would be obtained
> with a regular widefield epifluorescence microscope without confocal optics.
> >>
> >> 1. Can someone tell me at what confocal pinhole size this happens?
> >> 2. Can you also tell me where this was shown in the confocal literature
> for the first time (or a equivalently in a review article on same the
> topic)?
> >> 3. Does this principle also apply to spinning disk confocal
> microscopes, why or why not?
> >>
> >> Thank you greatly for your wisdom.
> >>
> >>
> >> John Oreopoulos
> >> Staff Scientist
> >> Spectral Applied Research Inc.
> >> A Division of Andor Technology
> >> Richmond Hill, Ontario
> >> Canada
> >> www.spectral.ca
> >
> >
> > ---------------------------------------------------------------------
> > *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.
> (fpc5p)
> > ---------------------------------------------------------------------
>

George McNamara

Re: At what confocal pinhole size does a confocal image cease to be confocal?

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

"pinhole size for different wavelengths,and why they were not bothered about differing section thickness. I was told by all that 'tests were performed on biological samples and it was found that in practice the majority of samples yielded very similar results when imaged with one versus multiple pinholes"

The sooner we all switch to STED, single molecule localization methods
(I'll include 3B, SOFI and similar methods with SML), dual view
isotropic DSLM/SPIM, or instant SIM, the sooner this pinhole diameter(s)
discussion will end - and more importantly, we will get better images.

some references:

dual view isotropic DSLM/SPIM eith joint 3D deconvolution =

Spatially isotropic four-dimensional imaging with dual-view plane
illumination microscopy. <http://www.ncbi.nlm.nih.gov/pubmed/24108093>

Wu Y, Wawrzusin P, Senseney J, Fischer RS, Christensen R, Santella A,
York AG, Winter PW, Waterman CM, Bao Z, Colón-Ramos DA, McAuliffe M,
*Shroff H*.

Nat Biotechnol. 2013 Nov;31(11):1032-8. doi: 10.1038/nbt.2713. Epub 2013
Oct 13.

PMID:
24108093

iSIM =

Instant super-resolution imaging in live cells and embryos via analog
image processing. <http://www.ncbi.nlm.nih.gov/pubmed/24097271>

York AG, Chandris P, Nogare DD, Head J, Wawrzusin P, Fischer RS, Chitnis
A, *Shroff H*.

Nat Methods. 2013 Nov;10(11):1122-6. doi: 10.1038/nmeth.2687. Epub 2013
Oct 6.

PMID:
24097271

Another use for R-L deconvolution: multi-resolution merging

Richardson-Lucy Deconvolution as a General Tool for Combining Images
with Complementary Strengths. <http://www.ncbi.nlm.nih.gov/pubmed/24436314>

Ingaramo M, York AG, Hoogendoorn E, Postma M, *Shroff H*, Patterson GH.

Chemphyschem. 2014 Jan 16. doi: 10.1002/cphc.201300831. [Epub ahead of
print]

PMID:
24436314

With respect to "Joint" data analysis (and no, I am not referring to
doing image processing in Colorado, where joints are now legal to buy),
I also encourage everyone to check out Figure 5 of:

Three-dimensional FRET reconstruction microscopy for analysis of dynamic
molecular interactions in live cells.
<http://www.ncbi.nlm.nih.gov/pubmed/18339754>

*Hoppe* AD, Shorte SL, *Swanson* JA, Heintzmann R.

Biophys J. 2008 Jul;95(1):400-18. doi: 10.1529/biophysj.107.125385. Epub
2008 Mar 13.

PMID:
18339754

raw vs "rec" (joint 3D deconvolution and spectral unmixing ... best to
just pay attention to the first 3 columns):
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2426648/figure/fig5/

enjoy,

George

On 1/30/2014 4:25 AM, phil laissue wrote:

> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> Hi John,
>
> it's looks bilinear, according to an earlier post by Brad Amos. See p 20
> here:
>
> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf
>
> On a related note, I had talked to several manufacturers a few years ago
> about this new trend of using one pinhole size for different wavelengths,
> and why they were not bothered about differing section thickness. I was
> told by all that 'tests were performed on biological samples and it was
> found that in practice the majority of samples yielded very similar results
> when imaged with one versus multiple pinholes', and the alignment of
> multiple pinholes was too much of a pain anyway. Having said that, some of
> the major manufacturers (sadly not all) have meanwhile implemented
> framescans with varying pinhole, which doesn't fully address the issue due
> to the time delay. An alternative would be a fast-switching pinhole for
> line scans, but I don't think anyone's looking into that.
>
> Cheers
>
> Philippe
>
>
>
>
> On 29 January 2014 17:19, John Oreopoulos<[hidden email]>wrote:
>
>
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>>
>> Hi Brian, yes I guess this gets back to the discussion I started a few
>> months back that led to an explanation about the differences between
>> "resolution" and "optical sectioning". My question to the listserver was
>> motivated by the observation that more often than not, one would like to
>> keep the specimen alive and use a little laser power as possible to image
>> the sample. A good way to achieve this is to turn down the laser power and
>> open up the confocal pinhole, thereby sacrificing some degree optical
>> sectioning. What surprises me is just how much one can actually open up the
>> pinhole sometimes and still get a "confocal enough" image that shows the
>> features of interest with good detail, and it is still far superior
>> compared to the widefield image. As you say, this is probably somewhat
>> dependent on the structure of interest in the cell/tissue that's being
>> imaged.
>>
>> Does anyone know if there is a way to express the loss of optical
>> sectioning ability as a function of pinhole size above 1 Airy unit? Can
>> this be quantified? Is it a linear trend?
>>
>> John
>>
>>
>> On 2014-01-29, at 11:59 AM, Armstrong, Brian wrote:
>>
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi John, for the theoretical limits of the gain in resolution you get
>>>
>> from using Confocal you could see Pawley's book ref mentioned previously.
>> For a practical limit; the gain from the Confocal is from the creation of
>> the optical slice through the use of a pinhole that excludes out of focus
>> light and therefore may increase the "resolution" of the image produced
>> (more signal / less noise). However, this depends upon the sample. If there
>> is no out of focus light beyond the range of the optical slice then there
>> is no gain in the Confocal image. Moreover, if your section is cut
>> physically thinner than the optical slice the widefield image and Confocal
>> image should be identical with 1 Airy unit pinhole size. In other words, it
>> wouldn't make much sense to use a Confocal microscope to image a 1um thick
>> tissue section (in most cases).
>>
>>> So, in my opinion, the answer is that it is largely sample dependent.
>>> *I imagine that you know this already.
>>> Cheers,
>>>
>>> Brian D Armstrong PhD
>>> Associate Research Professor
>>> Director, Light Microscopy Core
>>> Beckman Research Institute
>>> City of Hope
>>> Dept of Neuroscience
>>> 1450 E Duarte Rd
>>> Duarte, CA 91010
>>> 626-256-4673 x62872
>>>
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]]
>>>
>> On Behalf Of Guy Cox
>>
>>> Sent: Wednesday, January 29, 2014 5:59 AM
>>> To: [hidden email]
>>> Subject: Re: At what confocal pinhole size does a confocal image cease
>>>
>> to be confocal?
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> There is no one answer. A confocal microscope with a pinhole of
>>>
>> absolutely any size will provide more optical sectioning than a wide-field
>> microscope. The smaller the pinhole the better the optical sectioning.
>> However once one goes much below one Airy diameter the loss in signal will
>> pretty much negate any further gain. The question that has more relevance
>> is where can we get the sqrt2 increase in lateral resolution that confocal
>> potentially provides? The answer is that to get the whole value we need an
>> infinitely small pinhole, and one the pinhole reaches one Airy value the
>> resolution is the same as wide-field.
>>
>>> Guy Cox& Colin Sheppard, 2004. Practical limits of resolution in
>>>
>> confocal and non-linear microscopy. Microscopy Research& Technique, 63,
>> 18-22
>>
>>> I've really lost count of how many times I've posted this reference!
>>>
>>> Guy
>>>
>>> -----Original Message-----
>>> From: Confocal Microscopy List [mailto:[hidden email]]
>>>
>> On Behalf Of Turnbull, Lon
>>
>>> Sent: Wednesday, 29 January 2014 4:39 AM
>>> To: [hidden email]
>>> Subject: Re: At what confocal pinhole size does a confocal image cease
>>>
>> to be confocal?
>>
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>>
>>> Hi John,
>>>
>>> I do not know the answer to your first questions, but I can give you an
>>>
>> answer to your third question.
>>
>>> Spinning disk systems that I am familiar with have a fixed pinhole size
>>>
>> that is optimized for a particular objective, usually the highest power
>> objective. Therefore, the lower power objectives have a larger optical
>> section. With a small enough power objective, for example, the optical
>> section becomes very large and might even be considered to be non-confocal.
>> For example, if a 100x objective has a 0.91 micrometer optical section
>> then on the same system a 5x objective has a 95 micrometer optical section.
>>
>>>
>>> Lon
>>>
>>> Microscope manager
>>> Department of Biological Sciences
>>> University of North Texas
>>>
>>>
>>> Sent from my iPad
>>>
>>>
>>>> On Jan 28, 2014, at 11:04 AM, "John Oreopoulos"<
>>>>
>> [hidden email]> wrote:
>>
>>>> *****
>>>> To join, leave or search the confocal microscopy listserv, go to:
>>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>>> *****
>>>>
>>>> I'm wondering if the listserver can help me recall something I've heard
>>>>
>> several times before and point me to a reference that proves (and
>> demonstrates) it. Several people I think have said before (I'm looking in
>> the direction of Jim Pawley and Guy Cox perhaps) that beyond a certain
>> confocal pinhole size (in terms of Airy units), the image that is produced
>> by the confocal microscope is equivalent to that which would be obtained
>> with a regular widefield epifluorescence microscope without confocal optics.
>>
>>>> 1. Can someone tell me at what confocal pinhole size this happens?
>>>> 2. Can you also tell me where this was shown in the confocal literature
>>>>
>> for the first time (or a equivalently in a review article on same the
>> topic)?
>>
>>>> 3. Does this principle also apply to spinning disk confocal
>>>>
>> microscopes, why or why not?
>>
>>>> Thank you greatly for your wisdom.
>>>>
>>>>
>>>> John Oreopoulos
>>>> Staff Scientist
>>>> Spectral Applied Research Inc.
>>>> A Division of Andor Technology
>>>> Richmond Hill, Ontario
>>>> Canada
>>>> www.spectral.ca
>>>>
>>>
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>

--

George McNamara, Ph.D.
Single Cells Analyst
L.J.N. Cooper Lab
University of Texas M.D. Anderson Cancer Center
Houston, TX 77054
Tattletales http://works.bepress.com/gmcnamara/26/