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I have posted this before, but will repost.  We wrote a methods chapter on
spinning disk in 2003 (Maddox et al., methods in Enzymology) where we
discussed several of these issues.  I would be happy to provide a PDF for
those interested if you email me offline  ([log in to unmask]).

Happy imaging!

Paul


On 6/23/11 8:31 PM, "James Pawley" <[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
> *****
> 
> Well, what Lutz says is precisely true, but on
> the other hand, unless the stained part of the
> specimen is quite thick, it may not be very
> important.
> 
> Yes, light that originates far from the focus
> plane may come through the wrong pinhole. As the
> pinhole in an image plane it will actually arrive
> at the CCD in the wrong location. But on the
> other hand, it isn't very much light. In the
> Yoko, where the pinholes are 50µm and 250µm
> apart, the circle of transmission where the first
> light would come through has a circumference
> about 1570 µm (of which at most 6x50 or 300 µmm
> is open, more likely about 200 µm),  a diameter
> of 500 µm and an area of about 200k square
> microns. Of this, about 8000 square microns is
> "hole", about 4%. Apart from the geometry, the
> light focused back from the "spot" in the
> specimen tends to cluster near the pinhole, so
> the fraction of this out-of-focus light
> prohibited from reaching the detector is probably
> more than 96 parts out of 100.
> 
> It is then important to remember that this is a
> confocal microscope, and we often only get very
> few photons through the pinhole in any case
> (maybe tens-hundreds  in a 1 sec exposure) and "a
> lot less that very few is close to zero" if you
> will permit the imprecision.
> 
> The z-resolution of disc scanners was much
> studied when they first came out and the Z-image
> of a plane of fluorophor is almost the same as a
> single point scanner(with the same pinhole size)
> with the exception that when the focus plane
> moves far from focus, the signal level does not
> asymptote to zero, but to the amount of light
> that would pass an ND filter with the same
> average transmission as the disk. Other stain
> distributions may be produce other results (a
> periodic structure that appeared in the plane of
> the disk with a period that matched that of the
> hole pattern for instance) but it is not beyond
> the wit of man to obtain and use a measured 3D
> PSF to deconvolve confocal data of any type,
> including disc scanners.
> 
> I recommend it.
> 
> JP
> 
> 
>> *****
>> To join, leave or search the confocal microscopy listserv, go to:
>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>> *****
>> 
>> Hello,
>> 
>> it seems to me that you are dealing with the
>> common problem of pinhole cross talk, in which
>> the neighboring pinholes contribute in such way
>> that your PSF will contain next to the main peak
>> about six more of lesser intensity around it. To
>> deconvolve such image, you need this patterned
>> PSF! If you want to use a theoretical PSF you
>> need to work out the exact forward problem and
>> then generate a PSF that matches your instrument
>> in all parameters especially the geometry of the
>> cross-talking pinholes. Working with blind
>> deconvolution might be tricky, as there are
>> often heavy constraints on the PSF to introduce
>> additional knowledge. Unless your blind
>> deconvolution system supports such crosstalk
>> PSF's, I would not resort to this option.
>> Finally you can try to acquire an empirical PSF
>> using a small bead. The caveat is, that you can
>> only use a bead that is far enough away from
>> others so that it does not interfere with the
>> crosstalk pattern of other beads. Then, you just
>> cut out and center this single bead stack and
>> use it for deconvolution.
>> 
>> hope it helped
>> regards
>> Lutz
>> 
>> __________________________________
>> L u t z   S c h a e f e r
>> Sen. Scientist
>> Mathematical modeling / Image processing
>> Advanced Imaging Methodology Consultation
>> 16-715 Doon Village Rd.
>> Kitchener, ON, N2P 2A2, Canada
>> Phone/Fax: +1 519 894 8870
>> Email:     [log in to unmask]
>> ___________________________________
>> 
>> --------------------------------------------------
>> From: "Dani" <[log in to unmask]>
>> Sent: Thursday, June 23, 2011 10:22
>> To: <[log in to unmask]>
>> Subject: Deconvolving Spinning Disk Images
>> 
>>> *****
>>> To join, leave or search the confocal microscopy listserv, go to:
>>> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
>>> *****
>>> 
>>> Hi there,
>>> 
>>> I would like to deconvolve my spinning disk images for quantification
>>> purposes. Regarding this, I have a few issues/questions.
>>> 
>>> 1. I have been trying to image fluorescent beads, in order to obtain PSF
>>> measurements for my microscope (conditions). However, whenever I do this, I
>>> always "see the grid" of the spinning disk unit, meaning that there are
>>> multiple points visible at the same time, even when a single bead is imaged.
>>> This only becomes obvious after severe rescaling and the other grid dots
>>> have a very weak intensity compared to the main dot (at least 2 orders of
>>> magnitude). I have tried synchronizing imaging time with spinning disk
>>> speed, long/short exposures, high/low laser intensities, with/without EM
>>> gain, etc. I always see the same thing.
>>> The questions are: Will this affect the deconvolution efficiency/accuracy?
>>> Does anyone have any solution for this problem?
>>> 
>>> 
>>> 
>>> 2. I have read conflicting opinions on performing deconvolution using
>>> experimental vs. theoretical PSFs vs maximum likelihood estimations (MLE).
>>> Some say that when imaging thick samples (in my case ~30um) it is better to
>>> do MLEs, because the PSF changes depending on the depth that you image, and
>>> measured PSFs are always on the surface of the slide. Others say that
>>> measured PSFs will always (usually) be better as they don't assume anything
>>> but actually measure what is going on. Then again, I have been considering
>>> doing theoretical PSFs, as I have been having a lot of trouble actually
>>> measuring a proper PSF in my microscope (see point 1).
>>> Does anyone have any experience with this or can advise me on these issues.
>>> Which programs/algorithms/plugins could I best use for this?
>>> 
>>> 
>>> --
>>> View this message in context:
>>> http://confocal-microscopy-list.588098.n2.nabble.com/Deconvolving-Spinning-D
>>> isk-Images-tp6508446p6508446.html
>>> Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
> 
> 
> --
>                     ****************************************
> Prof. James B. Pawley,                                     Phone: 604-822-7801
> 3D Microscopy of Living Cells: Summer Course   CELL: 778-919-3176
> Info at: http://www.3dcourse.ubc.ca
> "If it isn't diffraction, it is statistics":Microscopist's complaint, Anon.