*****
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*****

Hi Kris

ABRF is marketing this workshop and study as 'trying to promote
reproducibility' however in the meantime there is a reproducibility issue
in the study itself that has dragged on for several months.   This issue
influences algorithm choice.  Respectfully, isn't it appropriate to push
hard to have ABRF solve this issue before doing a reproducibility workshop
next week?  If incorrect information, as to appropriate algorithm choice is
presented in the workshop or study it will take image processing
professionals their time to correct the mistaken impression.

I understand people at ABRF are busy and  I am absolutely not implying that
ABRF is trying to hide anything, or has intentionally done anything wrong
at all.  However, to be honest, I do not think ABRF (and many other
microscopy groups) fully appreciate the types of technical skills it takes
to do proper justice to complex digital image processing problems.
Oftentimes image processing workshops put on by microscopy groups are done
in too simple a manner and have led to a lack of rigor in image
processing.

Brian




On Tue, Apr 19, 2022 at 1:03 PM Kubow, Kristopher E - kubowke <
[log in to unmask]> wrote:

> *****
> To join or leave the confocal microscopy listserv or to change your email
> address, go to:
> https://lists.umn.edu/cgi-bin/wa?SUBED1=confocalmicroscopy&A=1
> Post images on http://www.imgur.com and include the link in your posting.
> *****
>
> Thanks for your input, Brian - it's much appreciated. And since a
> continued discussion of this sort is more appropriate for image.sc, let's
> move the conversation there. It is better to have the full threaded
> discussion in one place rather than cross-posted to the listserv.
>
> Also, I want to emphasize that the study is being conducted by a group of
> volunteers in our "spare time." All of us run core facilities - many of us
> completely by ourselves (no staff). Many of us also teach and/or have other
> obligations at our institutions. None of us are paid/compensated (either by
> our institutions or the ABRF) to run the study. We are in no way trying to
> hide anything. We are trying to do our best within our constraints. I
> understand you wish things would move more quickly (so do we!), but even
> tasks that might seem to be straightforward/quick/obvious/etc. do take time
> and require coordination among our members. I don't say this to be
> confrontational or condescending, but only to emphasize that our members
> are well-meaning, hard-working individuals who are trying to do their best
> with what they have.
>
> I look forward to engaging with you further on the deconvolution issue at
> image.sc.
>
> Best,
> -Kris
>
> ________________________________
> From: Brian Northan <[log in to unmask]>
> Sent: Monday, April 18, 2022 8:12 AM
> Subject: Re: 3D Image Analysis Tools and Reproducibility Event - April
> 27th via Zoom
>
> *****
> To join or leave the confocal microscopy listserv or to change your email
> address, go to:
>
> https://urldefense.proofpoint.com/v2/url?u=https-3A__lists.umn.edu_cgi-2Dbin_wa-3FSUBED1-3Dconfocalmicroscopy-26A-3D1&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=lMiutr1dy6jxfbZfb00NtOJHkGisOXchgySLZr_ltdM&s=qG6OzlR17xLhXVpo66_yZ3GUTqte32xrV4awF3aiY3k&e=
> Post images on
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> and include the link in your posting.
> *****
>
> Thanks Kris
>
> Just to re-emphasize why this issue is so important.  To get accurate
> intensities and segmentation of the nuclei images some type of image
> restoration must be done (either deconvolution or AI based restoration).
>  The segmentations I've seen of the nuclei image, so far, looked good at
> first glance (round well separated objects), however are merely
> simplifications which fit a spheroid to the data, but do not capture the
> true shape or intensities.
>
> Anyway, I will spend some time this week or next looking over the docker
> release and see if I can tell what happened with the PSF.  Seems like it is
> probably a resampling issue, perhaps even as simple as incorrect reading of
> spacings from the meta-data, but hard to know for sure.
>
> Brian
>
> Brian
>
> On Sun, Apr 17, 2022 at 8:08 PM Kubow, Kristopher E - kubowke <
> [log in to unmask]> wrote:
>
> > *****
> > To join or leave the confocal microscopy listserv or to change your email
> > address, go to:
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__lists.umn.edu_cgi-2Dbin_wa-3FSUBED1-3Dconfocalmicroscopy-26A-3D1&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=lMiutr1dy6jxfbZfb00NtOJHkGisOXchgySLZr_ltdM&s=qG6OzlR17xLhXVpo66_yZ3GUTqte32xrV4awF3aiY3k&e=
> > Post images on
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=lMiutr1dy6jxfbZfb00NtOJHkGisOXchgySLZr_ltdM&s=tCrWXALdZYlJ-BHrrWfqDrabDddXGFqbajpsBYTjJN0&e=
> and include the link in your posting.
> > *****
> >
> > Question was cross-posted on image.sc forum please see forum for
> response
> > and continuation of discussion:
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__forum.image.sc_t_3d-2Dimage-2Danalysis-2Dtools-2Dand-2Dreproducibility-2Devent-2Dapril-2D27th-2Dvia-2Dzoom_64671_5-3Fu-3Dkris-5Fkubow&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=lMiutr1dy6jxfbZfb00NtOJHkGisOXchgySLZr_ltdM&s=-Y-XonaNJ5KjpvnIEH1yhfrJANHJuRnQqU15_lCOqxs&e=
> >
> > ________________________________
> > From: Brian Northan <[log in to unmask]>
> > Sent: Friday, April 15, 2022 6:09 AM
> > Subject: Re: 3D Image Analysis Tools and Reproducibility Event - April
> > 27th via Zoom
> >
> > *****
> > To join or leave the confocal microscopy listserv or to change your email
> > address, go to:
> >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__lists.umn.edu_cgi-2Dbin_wa-3FSUBED1-3Dconfocalmicroscopy-26A-3D1&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=hBsapqAzr5hAx-G9-qM3LSc9TEwTGgBoh0n4rbwkBks&s=z0i7P1WXTT-6ZCuw6KqHreKuHf-jiYoJTFfEJo1rass&e=
> > Post images on
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=hBsapqAzr5hAx-G9-qM3LSc9TEwTGgBoh0n4rbwkBks&s=wPbLmuOcWhpfciV0CceO3Wq0xE9rQgqBl5U33Gian8E&e=
> > and include the link in your posting.
> > *****
> >
> > Hi Kris
> >
> > Any update on this? Why not just release the forward model code
> publically
> > and have the community look it over?  I don't understand how ABRF can
> claim
> > they are addressing 'reproducibility' when they are working in such a
> > closed manner.  Isn't openness and communication the number one principle
> > of reproducibility?
> >
> > Brian
> >
> > On Thu, Mar 24, 2022 at 3:28 PM Kubow, Kristopher E - kubowke <
> > [log in to unmask]> wrote:
> >
> > > *****
> > > To join or leave the confocal microscopy listserv or to change your
> email
> > > address, go to:
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__lists.umn.edu_cgi-2Dbin_wa-3FSUBED1-3Dconfocalmicroscopy-26A-3D1&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=hBsapqAzr5hAx-G9-qM3LSc9TEwTGgBoh0n4rbwkBks&s=z0i7P1WXTT-6ZCuw6KqHreKuHf-jiYoJTFfEJo1rass&e=
> > > Post images on
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=hBsapqAzr5hAx-G9-qM3LSc9TEwTGgBoh0n4rbwkBks&s=wPbLmuOcWhpfciV0CceO3Wq0xE9rQgqBl5U33Gian8E&e=
> > and include the link in your posting.
> > > *****
> > >
> > > Thanks Brian and David. We agree that there does seem to be a mismatch
> > > between the spacing parameters that were given and what is actually
> used
> > in
> > > the nuclei images. This will impact deconvolution of the images, if
> > people
> > > elect to do that as part of their segmentation protocol for the study.
> > > We’re currently looking into the problem and are in contact with the
> > group
> > > that developed the simulation software. We'll post updates (when we get
> > > them) on the image.sc forum:
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__forum.image.sc_t_3d-2Dimage-2Danalysis-2Dtools-2Dand-2Dreproducibility-2Devent-2Dapril-2D27th-2Dvia-2Dzoom_64671&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=hBsapqAzr5hAx-G9-qM3LSc9TEwTGgBoh0n4rbwkBks&s=SMse04bgkg546B_NFjehOds-hRMIW4bovk4mbCxMeqc&e=
> > >
> > > ________________________________
> > > From: Brian Northan <[log in to unmask]>
> > > Sent: Wednesday, March 23, 2022 9:26 AM
> > > Subject: Re: 3D Image Analysis Tools and Reproducibility Event - April
> > > 27th via Zoom
> > >
> > > *****
> > > To join or leave the confocal microscopy listserv or to change your
> email
> > > address, go to:
> > >
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__lists.umn.edu_cgi-2Dbin_wa-3FSUBED1-3Dconfocalmicroscopy-26A-3D1&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=Bbo8EpRKzdBsReltSfbg8Udr6_-vbdBTWzo2_VmZUow&s=-owGyRKzOtmTtqOXx9hHPa81IsG5cCt5iCVjWV4ILTg&e=
> > > Post images on
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=Bbo8EpRKzdBsReltSfbg8Udr6_-vbdBTWzo2_VmZUow&s=uQ0i7zYMybxCqH94JEtgiMLq5Tqf4RT45jB8kGVrshU&e=
> > > and include the link in your posting.
> > > *****
> > >
> > > Interestingly I used similar spacings to those David estimated.  After
> > > first using the given spacings and getting terrible deconvolution
> > results,
> > > I simply used the Nyquist spacings and got a much better result.
> > > From there I tweaked the spacings a bit and used 0.1 XY and 0.8 Z and
> > got a
> > > pretty good deconvolution.
> > >
> > > I mentioned this to Kris Kublow, one of the organizers several months
> ago
> > > and he said the group would look into it, but I never heard if they
> did.
> > >
> > > Jessica, did your group ever run tests to validate the PSF, forward
> model
> > > and spacings?  If so, how did you convince yourself the simulation was
> > > correct?
> > >
> > > Brian
> > >
> > >
> > > On Mon, Mar 21, 2022 at 1:09 PM David Biggs <[log in to unmask]>
> wrote:
> > >
> > > > *****
> > > > To join or leave the confocal microscopy listserv or to change your
> > email
> > > > address, go to:
> > > >
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__lists.umn.edu_cgi-2Dbin_wa-3FSUBED1-3Dconfocalmicroscopy-26A-3D1&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=Bbo8EpRKzdBsReltSfbg8Udr6_-vbdBTWzo2_VmZUow&s=-owGyRKzOtmTtqOXx9hHPa81IsG5cCt5iCVjWV4ILTg&e=
> > > > Post images on
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=Bbo8EpRKzdBsReltSfbg8Udr6_-vbdBTWzo2_VmZUow&s=uQ0i7zYMybxCqH94JEtgiMLq5Tqf4RT45jB8kGVrshU&e=
> > > and include the link in your posting.
> > > > *****
> > > >
> > > > In reference to Brian's observations, I also concur that the
> parameters
> > > > provided for the widefield nuclei dataset do not match the data
> > provided.
> > > > For a 0.75 NA air lens, I estimate the Z spacing is closer to 0.75um,
> > and
> > > > the XY spacing is closer to 0.1um.
> > > > I roughly estimated these from the shape of the missing cone in the
> > > Fourier
> > > > domain.
> > > >
> > > > Any clarification would be appreciated.
> > > >
> > > > Thanks,
> > > > David
> > > >
> > > >
> > > >
> > > > On Sat, Mar 19, 2022 at 1:44 PM Brian Northan <[log in to unmask]>
> > > wrote:
> > > >
> > > > > *****
> > > > > To join or leave the confocal microscopy listserv or to change your
> > > email
> > > > > address, go to:
> > > > >
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__lists.umn.edu_cgi-2Dbin_wa-3FSUBED1-3Dconfocalmicroscopy-26A-3D1&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=Bbo8EpRKzdBsReltSfbg8Udr6_-vbdBTWzo2_VmZUow&s=-owGyRKzOtmTtqOXx9hHPa81IsG5cCt5iCVjWV4ILTg&e=
> > > > > Post images on
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=Bbo8EpRKzdBsReltSfbg8Udr6_-vbdBTWzo2_VmZUow&s=uQ0i7zYMybxCqH94JEtgiMLq5Tqf4RT45jB8kGVrshU&e=
> > > and include the link in your
> > > > posting.
> > > > > *****
> > > > >
> > > > > Has anyone else tried to deconvolve the nuclei images used in the
> > > > study?  I
> > > > > was only able to obtain a good deconvolution result after
> > introducing a
> > > > > fudge factor into the PSF generation.  Is anybody else running into
> > the
> > > > > same problem?  My concern is that there is a potential sampling
> issue
> > > in
> > > > > the PSF used in the simulation, and that in turn could (falsely)
> make
> > > it
> > > > > seem as if deconvolution doesn't work.  That would be a shame,
> > > > > deconvolution is an important tool for processing 3D Widefield
> > > > fluorescence
> > > > > images, and it would be nice if this study could be used, to
> > > demonstrate
> > > > > the benefits of deconvolution.
> > > > >
> > > > > Contact me off list if you would like.  I'd like to verify that
> these
> > > > > images can be deconvolved using standard tools.
> > > > >
> > > > > Brian
> > > > >
> > > > > On Fri, Mar 18, 2022 at 9:44 AM Jessica Hornick <
> > > > > [log in to unmask]>
> > > > > wrote:
> > > > >
> > > > > > *****
> > > > > > To join or leave the confocal microscopy listserv or to change
> your
> > > > email
> > > > > > address, go to:
> > > > > >
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__lists.umn.edu_cgi-2Dbin_wa-3FSUBED1-3Dconfocalmicroscopy-26A-3D1&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=Bbo8EpRKzdBsReltSfbg8Udr6_-vbdBTWzo2_VmZUow&s=-owGyRKzOtmTtqOXx9hHPa81IsG5cCt5iCVjWV4ILTg&e=
> > > > > > Post images on
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=http-3A__www.imgur.com&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=Bbo8EpRKzdBsReltSfbg8Udr6_-vbdBTWzo2_VmZUow&s=uQ0i7zYMybxCqH94JEtgiMLq5Tqf4RT45jB8kGVrshU&e=
> > > and include the link in your
> > > > > posting.
> > > > > > *****
> > > > > >
> > > > > > Come learn more about 3D image analysis tools and how they can
> help
> > > > > > improve reproducibility at this two-hour online workshop hosted
> by
> > > the
> > > > > > Light Microscopy Research Group of the ABRF!
> > > > > >
> > > > > > This event will be centered around the LMRG’s ongoing study of
> > > > > > reproducibility in 3D image analysis in which we seek to
> > characterize
> > > > and
> > > > > > identify sources of (ir)reproducibility in 3D segmentation. Try
> > your
> > > > hand
> > > > > > at analyzing the study images and then attend the workshop to see
> > how
> > > > the
> > > > > > pros would do it. Representatives from several major 3D analysis
> > > > > platforms
> > > > > > will walk attendees through how to segment and analyze the images
> > > from
> > > > > our
> > > > > > study and will discuss how their platforms support reproducible
> > image
> > > > > > analysis. These demonstrations will occur simultaneously in
> > breakout
> > > > > rooms
> > > > > > and attendees may choose up to two to attend. There will also be
> an
> > > > open
> > > > > > session at the conclusion of the program during which attendees
> may
> > > > visit
> > > > > > and talk with any representative they like. Note that the
> platforms
> > > > will
> > > > > > not be competing against each other–the purposes of the event are
> > > > > > instructional/informational and to promote reproducibility in
> image
> > > > > > analysis.
> > > > > >
> > > > > > We will host representatives from the following platforms:
> > > ImageJ/FIJI,
> > > > > > Napari, CellProfiler, Arivis, and Volocity. We hope to add more
> to
> > > this
> > > > > > list before the event.
> > > > > >
> > > > > > The event will take place on April 27th at 8:00 am Pacific Time /
> > > 11:00
> > > > > am
> > > > > > Eastern Time via Zoom.
> > > > > >
> > > > > > ** Register for the event here:
> > > > > >
> > > > >
> > > >
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__us02web.zoom.us_meeting_register_tZEsceuvpzIvH9e0kFHEuWdxRMBAgrrpUwzj&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=Bbo8EpRKzdBsReltSfbg8Udr6_-vbdBTWzo2_VmZUow&s=df5s8P1S7R061gnHC6OXrqAOlhVJ3FaJP5Wt8KsC0Qo&e=
> > > > > >
> > > > > > This event is targeted to people who are interested in learning
> > more
> > > > > about
> > > > > > a specific image analysis platform, learning how to use an image
> > > > analysis
> > > > > > tool better, and/or learning about how reproducibility is
> addressed
> > > by
> > > > > > these platforms.
> > > > > >
> > > > > > We strongly encourage attendees to participate in the study
> before
> > > the
> > > > > > event so that you are familiar with the image analysis problem.
> > > > Attendees
> > > > > > who participate in the study before the event will be entered in
> a
> > > $50
> > > > > > Amazon gift card drawing. (If you’ve already participated in the
> > > study
> > > > > and
> > > > > > didn’t win in our last drawing, you’re automatically entered.)
> > Study
> > > > info
> > > > > > can be found here:
> > > > >
> > >
> >
> https://urldefense.proofpoint.com/v2/url?u=https-3A__sites.google.com_view_lmrg-2Dimage-2Danalysis-2Dstudy&d=DwIFaQ&c=eLbWYnpnzycBCgmb7vCI4uqNEB9RSjOdn_5nBEmmeq0&r=wgGHXpNkPeE6PJJsl0IvoA&m=Bbo8EpRKzdBsReltSfbg8Udr6_-vbdBTWzo2_VmZUow&s=AvCI1kJRHZzgNZUP-7MfZJdOZZaCKUJOvPZAamulY-0&e=
> > > > > >
> > > > > > If you have questions about the event, please contact Jessica
> > > Hornick (
> > > > > > [log in to unmask]).
> > > > > >
> > > > >
> > > >
> > >
> >
>