*****
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:[log in to unmask]] On Behalf Of Guy Cox
>>> Sent: Wednesday, January 29, 2014 5:59 AM
>>> To: [log in to unmask]
>>> 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:[log in to unmask]] On Behalf Of Turnbull, Lon
>>> Sent: Wednesday, 29 January 2014 4:39 AM
>>> To: [log in to unmask]
>>> 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" <[log in to unmask]> 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)
>>> ---------------------------------------------------------------------