Thank you all for the responses.  I guess what I thought was simple was not the case.

As I had not done the experiments, and when I explain our setup, you’ll see our limitations, and what were some great take home points.

We cannot put a pinhole in front of our non-descanned detector (maybe we could, but it would not be trivial), and we do not have any descanned detectors on this system (it is MP only).
An additional IR block in front of the detector may be useful.
Broadband excitation of multiple flours needs to be considered.  We can modify filters here.
I had no idea you approached WF psf so fast as you open a pinhole past 1 Airy.
In xy, the psf between 2P and 1P will be relatively equivalent, but not so in z.

If I am wrong on any of these points, I would gratefully like to know, off list.

Thanks so much.


Best,

Gary



Gary Laevsky, Ph.D.
Confocal Imaging Facility Manager
Dept. of Molecular Biology
Washington Rd.
Princeton University 
Princeton, New Jersey, 08544-1014
(O) 609 258 5432
(C) 508 507 1310

On Nov 18, 2013, at 9:48 AM, Jeff Reece <[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 think Figure 4 in the Amos et al chapter is a great way to visualize how the excitation psf dominates the overall confocal psf for xy resolution.
> Similar figures for z resolution are what we need to answer the original question, comparing confocal with 2P.  So there would be Fig 4z-conf and Fig 4z-2P.  Fig Fig 4z-conf would also demonstrate the utility of the pinhole in z compared to confocal xy.  I cannot quickly create these myself right now but will describe them.
> 
> In both Figs 4z-conf and 4z-2P, the first row showing the graphs of the pinhole dimensions would be eliminated.  In Fig 4z-conf, one might instead replace it with statements of the pinhole size in AU used for that column.
> 
> In Fig 4z-conf, the roles of excitation psf (2nd row of Fig 4) and emission psf (3rd row of Fig 4) are reversed, in that the emission psf will dominate (be smaller than) the overall psf.  When the pinhole is completely closed, the emission psf will typically be much smaller than the excitation psf.  As the pinhole is opened, the emission psf will gradually get as big as the excitation psf, or more accurately, as big as the standard widefield emission psf, which is usually slightly bigger than the excitation psf.
> 
> In Fig 4z-2P, it might be useful to add another row before the 2P excitation psf that shows the 1P version of the psf, at the 2P excitation wavelength, i.e. before the 2P effect occurs, so one can visualize the effect of squaring the 1P psf to get the true 2P excitation psf.
> The 2P emission psf would be the WF emission psf equivalent to the open pinhole in confocal mode.  The 2P excitation psf would dominate the overall 2P psf, as in the case for confocal xy.
> 
> Probably clear as mud without the real figure.
> 
> -Jeff
> 
> 
> 
> 
>> ________________________________
>> From: Steffen Dietzel <[log in to unmask]>
>> To: [log in to unmask] 
>> Sent: Monday, November 18, 2013 5:47 AM
>> Subject: Re: 2P vs 1P psf
>> 
>> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> Citable literature is sparse in that area. For two-photon and confocal 
>> resolution formula I usually cite these two, the first one being the one 
>> that Guy already mentioned:
>> 
>> G. Cox, C. J. Sheppard: Practical limits of resolution in confocal and 
>> non-linear microscopy. In: Microscopy Research and Technique. Band 63, 
>> Issue 1, 2004, p. 18–22, doi:10.1002/jemt.10423, PMID 14677129.
>> 
>> B. Amos, G. McConnell, T. Wilson: Confocal microscopy. In: E. Egelman 
>> (Ed.): Biophysical Techniques for Characterization of Cells (= 
>> Comprehensive Biophysics. 2). Elsevier, Academic Press, Amsterdam 2012, 
>> ISBN 978-0-12-374920-8, 2, p. 3–23, doi:10.1016/B978-0-12-374920-8.00203-4
>> Author version:
>> http://www2.mrc-lmb.cam.ac.uk/images/groupleaders/Confocal_microscopy_Amos_McConnell_Wilson.pdf
>> 
>> 
>> When it comes to three-photon excition it gets really thin. I just very 
>> recently came accross a paper which gives the xy-resolution formula 
>> (0.51*lambda)/(sqrt(3)*NA), see below. If anyone should have an older 
>> citable reference. I very much would like to hear about it.
>> 
>> Steffen
>> 
>> Jung Ho Yu, Seung-Hae Kwon, Zdenˇek Petrášek3, Ok Kyu Park, Samuel 
>> Woojoo Jun, Kwangsoo Shin, Moonkee Choi, Yong Il Park, Kyeongsoon Park, 
>> Hyon Bin Na, Nohyun Lee, Dong Won Lee, Jeong Hyun Kim, Petra Schwille, 
>> Taeghwan Hyeon: High-resolution three-photon biomedical imaging using 
>> doped ZnS nanocrystals. In: Nature Materials. 12, 2013, p. 359–366, 
>> doi:10.1038/NMAT3565
>> 
>> 
>> On 17.11.2013 16:06, Jeff Reece wrote:
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>> 
>>> Is there a reference for the confocal psf being equal to the excitation psf when the pinhole=1AU (free access via the web is requested).  It is not intuitive that the confocal psf would leave out the optics (pinhole) of the emission path.
>>> 
>>> Thanks,
>>> Jeff
>>> 
>> 
>> 
>> -- 
>> ------------------------------------------------------------
>> Steffen Dietzel, PD Dr. rer. nat
>> Ludwig-Maximilians-Universität München
>> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
>> Head of light microscopy
>> 
>> Mail room:
>> Marchioninistr. 15, D-81377 München
>> 
>> Building location:
>> Marchioninistr. 27,  München-Großhadern
>> 
>> 
>> 
>>