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"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<[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
>> *****
>>
>> 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
>>>>          
>>>
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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/