Posted by
James Pawley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolving-Spinning-Disk-Images-tp6508446p6510509.html
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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
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>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:
[hidden email]
>___________________________________
>
>--------------------------------------------------
>From: "Dani" <
[hidden email]>
>Sent: Thursday, June 23, 2011 10:22
>To: <
[hidden email]>
>Subject: Deconvolving Spinning Disk Images
>
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>>
>>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:
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http://confocal-microscopy-list.588098.n2.nabble.com/Deconvolving-Spinning-Disk-Images-tp6508446p6508446.html>>Sent from the Confocal Microscopy List mailing list archive at Nabble.com.
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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.