Posted by
Lutz Schaefer on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolving-Spinning-Disk-Images-tp6508446p6510267.html
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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:
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From: "Dani" <
[hidden email]>
Sent: Thursday, June 23, 2011 10:22
To: <
[hidden email]>
Subject: Deconvolving Spinning Disk Images
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>
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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?
>
>
> --
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>
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