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David,
the Zeiss ZEN iterative deconvolution algorithms use the missing data
facility. It is activated simply by input pixels that have zero grey value
and by setting the first estimate to the mean of the input image (selection
via the advanced settings). It is important to consider that "missing" data
is actually contained within the PSF support. Therefore, the region or
volume of zero values should not be larger than the PSF. Hope that helps.
Regards
Lutz
__________________________________
L u t z S c h a e f e r
Sen. Scientist
Mathematical modeling / Computational microscopy
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]
___________________________________
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From: "David Baddeley" <[log in to unmask]>
Sent: Monday, November 12, 2012 15:25
To: <[log in to unmask]>
Subject: Re: Deconvolution question
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I'd agree with Ryan in saying that, unless saturated the bright objects
> should be left in, and that any resulting artifacts would be a sign that
> something else was wrong with the deconvolution process (my money would be
> on the PSF measurement). If the user has saturated the bright objects,
> however, then the image will not be able to be deconvolved in a straight
> forward manner.
>
> Some deconvolution algorithms can deal with 'missing' data, ignoring
> certain pixels and reconstructing the image on the remainder, and this
> might be an option for dealing with saturated data if the software
> supports it (would any vendors care to comment here - is there a facility
> for specifying invalid pixels in any of the commercial packages?). I've
> done this quite successfully in the past with a custom variant of the ICTM
> algorithm in which the misfit on the invalid pixels is set to zero before
> being back-propagated. The missing data facility, in combination with
> padding, is used behind the scenes in practically all deconvolution
> algorithms to suppress edge effects, so it's mostly a question of whether
> this is exposed to the user in any form.
>
> cheers,
> David
>
>
>
> ________________________________
> From: "Ryan, Kevin" <[log in to unmask]>
> To: [log in to unmask]
> Sent: Friday, 9 November 2012 10:08 AM
> Subject: Re: Deconvolution question
>
> *****
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>
> (Commercial producer of AutoQuant, but this particular answer is for any
> deconvolution system)
>
> The PSF of an optical system is modeled as (and deconvolved as) a linear
> shift invariant (LSI) system. Non-linear changes, such as zeroing out
> inconvenient data prior to deconvolution, will give you a bad
> deconvolution in any region (above, below, or near in XY) within the range
> of the PSF. That's because you will have a dimmer, unclipped remainder of
> the blob unsupported by a PSF, and you will have objects outside the blob
> that are missing their PSF extent through the clipping region. Not to
> mention the sharp edges of the cut region - those will indeed be likely to
> come through the deconvolution amplified to some extent. A sharp-edged
> clip is the worst possible method, introducing lots of high spatial
> frequency noise; a Gaussian dimming of the blob would be better, but more
> complex, and still a non-linear and shift variant change to the
> observations.
>
> [ Linear changes mean that Ax + Bx = (A+B)x, that the observed data can be
> treated as a simple sum of the PSF's of each fluorescent point in the
> original object; shift invariant means that the same PSF applies all
> through the objects ]
>
> Unless the bright objects are _saturating_ your camera/detector, leave
> them in. Perform the deconvolution and then, only then, clip the bright
> fluorescent blobs from the reconstruction. They'll be considerably smaller
> after deconvolution, non-linear clipping won't affect anything outside
> those regions, and it will be easier to adjust contrast for the objects of
> interest without them.
>
> If your user is saturating his images, that itself is a non-linear change
> of your observations - and may well be the source of his deconvolution
> issues. Have the user reduce exposure times until his observations are no
> longer saturated. If that is not workable (perhaps the features of
> interest become too dim, get lost in noise or quantization binning), I
> would suggest a higher bit depth camera.
>
> Otherwise, your user is going to have to live with some level of
> distortion in their deconvolution reconstructions.
>
>
> Kevin Ryan
> Media Cybernetics, Inc.
>
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Kate Luby-Phelps
> Sent: Thursday, November 08, 2012 12:16 PM
> To: [log in to unmask]
> Subject: Deconvolution question
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> I have a user who has unavoidable brightly fluorescent blobs in his field
> of view that interfere with deconvolution of the less fluorescent
> structures. I just found out he has been masking them out before
> deconvolving by making isosurface objects and setting the voxels inside
> the object to zero. From what I understand about deconvolution, this seems
> like a bad idea because it introduces (a) void(s) with sharp edges in the
> dataset as well as clipping out information that likely came from regions
> of the specimen outside the blob. I think he should crop the z stack to
> exclude the blobs and then deconvolve but I don't know how important this
> issue actually is. Can anyone advise me on this?
>
> Thanks
>
> Kate Luby-Phelps
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