Dear Vincent,
I disagree, taking 3 planes will not permit calculation of a single
plane in 3D unless there is no signal outside the measured planes! Just
3 planes is too sparse when signal originates from outside the plane of
interest. Taking the 2 extra planes can be used to reduce blur (i.e.
increase contrast) but is is not deconvolution.
Hope this helps
Mark Cannell
Vincent wrote:
> Hi Jan,
>
> Just to make things clear, I would like to add to this that the 2D deconv. is
> aadvised for WF images.
> If you want a single confocal plane, we advise to image at least an adjacent
> plane on both sides, before performing (3D) deconvolution.
>
> Best regards,
> Vincent
>
>
>
> Jan Trnka wrote:
> > Thanks for all the comments. What would be the advantages of using WF over
> > wide-pinhole confocal (assuming it's the same microscope)?
> >
> > Off list Vincent suggested using 2D deconv. with a theoretical PSF, which
> > might be the way to go. It's my understanding that deconvolution has a place
> > in WF imaging (which I guess is close to what I'm doing), am I correct?
> >
> > Thanks again,
> >
> > Jan
> >
> > On 12 Aug, 2010, at 3:23 PM, Guy Cox wrote:
> >
> >> Well, I’m not clear why you are using confocal at all for this application.
> >> If Z resolution doesn’t matter surely you will be better in wide field? And
> >> I really can’t see how deconvolution will help. /Pace/ various vendors, if
> >> you cannot sample at the resolution limit, I don’t think it will give you any
> >> more than conventional noise-reduction and sharpening filters.
> >>
> >>
> >> Guy
> >>
> >> Optical Imaging Techniques in Cell Biology
> >> by Guy Cox CRC Press / Taylor & Francis
> >> http://www.guycox.com/optical.htm
> >> ______________________________________________
> >> Associate Professor Guy Cox, MA, DPhil(Oxon)
> >> Australian Centre for Microscopy & Microanalysis,
> >> Madsen Building F09, University of Sydney, NSW 2006
> >>
> >> Phone +61 2 9351 3176 Fax +61 2 9351 7682
> >> Mobile 0413 281 861
> >> ______________________________________________
> >> http://www.guycox.net
> >>
> >>
> >> *From:* Confocal Microscopy List
> >> [mailto:[log in to unmask]] *On Behalf Of *Jan Trnka
> >> *Sent:* Thursday, 12 August 2010 10:49 PM
> >> *To:* [log in to unmask] <mailto:[log in to unmask]>
> >> *Subject:* Re: Optical slice thickness and number for PSF and deconvolution
> >>
> >> OK, I understand that it's best to sample at Nyquist. However, for my
> >> experimental samples (looking at mitochondria in live cells over several
> >> hours) this would cause a lot of photodamage and bleaching. Since I don't
> >> really care about z-resolution (at this point I basically want a 2D picture
> >> of a whole cell with all its mitochondria) I decided to go for a wide
> >> pinhole, which allows me to get the picture I want in one go. Maybe this
> >> isn't the best way of doing it (any suggestions welcome) but my impression is
> >> that such a picture (or a z-stack of thick slices) could be deconvolved to
> >> get sharper pictures. Does this make sense? If so, how do I get a PSF for
> >> such deconvolution?
> >>
> >> Jan
> >>
> >> On 12 Aug, 2010, at 2:12 PM, Vincent wrote:
> >>
> >>
> >> *commercial interest*
> >>
> >> Dear Jan,
> >>
> >> The amount of the signal in images is mostly judged just after image
> >> acquisition. Based on this it is often decided to use a wider pinhole.
> >> As you probably know, when deconvolution is properly performed you will gain
> >> not only an increase in resolution but also in signal. Therefore, we advise
> >> to close the pinhole and use deconvolution for increasing the signal (to
> >> noise) before determining the quality of the image.
> >>
> >> As with imaging the object of interest it is important to follow the Nyquist
> >> criteria for imaging the bead images.
> >> We have a Nyquist calculator on our website (www.svi.nl/NyquistCalculator
> >> <http://www.svi.nl/NyquistCalculator>) to determine these rates. You can also
> >> create a picture here of your theoretical PSF to get an idea of its dimensions.
> >>
> >> In general it is best to really match the Nyquist criterion in xyz. Else you
> >> can go for 2x more. This however may introduce other problems like e.g.,
> >> bleaching. If the bead images are differently sampled it requires
> >> interpolation for matching that, making the process of deconvolution more
> >> computationally demanding. Thus Nyquist is okay. Another important thing to
> >> keep in mind is that you need to image enough planes to cover your PSF.
> >>
> >> I hope this answers your questions.
> >> Best regards,
> >> Vincent
> >>
> >> ***********************************************************
> >> Vincent Schoonderwoert, PhD
> >> Scientific Volume Imaging bv
> >> Hilversum, The Netherlands
> >> [log in to unmask] <mailto:[log in to unmask]>
> >> [log in to unmask] <mailto:[log in to unmask]>
> >> Tel: + 31 35 646 8216
> >> ***********************************************************
> >>
> >>
> >>
> >>
> >> Jan Trnka wrote:
> >> Dear list,
> >>
> >> this is probably a trivial question but so far I haven't found a good answer.
> >> When taking 3D images of subresolution beads in a confocal microscope (for
> >> PSF construction) does the number and thickness of slices in the z-stack need
> >> to be exactly the same as that of a sample to be deconvolved? I understand
> >> the x-y dimensions need to be the same but how does it work for z? Would a
> >> higher number of thinner slices (finer z resolution) of the bead improve the
> >> construction of the PSF? My actual samples are imaged with a rather wide
> >> pinhole setting to limit the exposure of the sample (live cells) and thus
> >> provide quite thick optical sections.
> >>
> >> Thanks,
> >>
> >> Jan
> >>
> >> Jan Trnka, MD, PhD
> >> Department of Biochemistry
> >> 3rd Medical Faculty
> >> Ruska 87
> >> 100 00 Praha 10
> >> Czech Republic
> >> [log in to unmask] <mailto:[log in to unmask]>
> >> Tel.: +420 26710 2410
> >>
> >>
> >>
> >>
> >>
> >>
> >>
> >>
> >> Jan Trnka, MD, PhD
> >> Department of Biochemistry
> >> 3rd Medical Faculty
> >> Ruska 87
> >> 100 00 Praha 10
> >> Czech Republic
> >> [log in to unmask] <mailto:[log in to unmask]>
> >> Tel.: +420 26710 2410
> >>
> >>
> >>
> >>
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> >
> > Jan Trnka, MD, PhD
> > Department of Biochemistry
> > 3rd Medical Faculty
> > Ruska 87
> > 100 00 Praha 10
> > Czech Republic
> > [log in to unmask] <mailto:[log in to unmask]>
> > Tel.: +420 26710 2410
> >
> >
> >
>
>
> --
> ***********************************************************
> Vincent Schoonderwoert, PhD
> Imaging Specialist/Account Manager
> [log in to unmask]
>
> Scientific Volume Imaging bv
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