Dear Jan,

I absolutely agree with Sudipta: (1) use the same pinhole to measure PSF as you use for imaging, (2) extra resolution in acquiring the PSF won't hurt. 
There are many other considerations, of course, for example: PSF at the edge of the field is usually wider than in the center; if the cells are thick, then spherical aberration may strongly affect the PSF at deeper layers, and you might get better results by using a theoretical PSF that takes spherical aberration into account than with experimental PSF; it also helps to remember about axial scaling...

Mike Model


________________________________________
From: Confocal Microscopy List [[log in to unmask]] On Behalf Of Sudipta Maiti [[log in to unmask]]
Sent: Saturday, August 14, 2010 12:36 PM
To: [log in to unmask]
Subject: Re: Optical slice thickness and number for PSF and deconvolution

Wait a minute. Since Mark and Guy are involved, there is something useful to
be learned here, and I was reading this with intent. But the original
question had two points that I feel were not adequately addressed by either.
First, the number of Z-slices required for imaging the bead: more than the
original can help, as the deconvolution algorithm will probably use a smooth
function to model the PSF obtained from the subresolution bead image, and use
that for deconvolution.
Second, was there something about using a larger pinhole during the actual
image acquisition? The pinhole size should match for the actual imaging and
the bead - otherwise you don't get the same PSF. I guess it is possible to
calculte the PSF for other pinhole sizes, but may not be the best thing to do.
Sudipta

    On Sat, 14 Aug 2010 23:02:18 +1000, Guy Cox wrote
> It's a bit challenging to disagree with Mark but .... we are interested
> in signal over noise.  Opening the pinhole beyond the diameter of the
> Airy disk will let in a little more signal (from the outer rings)
>  and a LOT more noise.  In almost every case it will make things worse.
> Photons are precious - if they come from where we want - other
> photons are something we need to exclude at all costs.
>
> As to the question about 3 sections - Mark is quite right, of course,
>  if we are dealing with a thick sample, but if I've followed this thread
> correctly we are dealing with thin cells where the information is
> largely in one plane.  If this is so we should be able to do pretty good
> deconvolution with 3 sections.
>
>                                                       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
>
> -----Original Message-----
> From: Confocal Microscopy List [mailto:[log in to unmask]]
> On Behalf Of Mark Cannell
> Sent: Friday, 13 August 2010 8:52 AM
> To: [log in to unmask]
> Subject: Re: Optical slice thickness and number for PSF and
> deconvolution
>
> Hi All
>
> I'm sorry but this advice is wrong. The pinhole is a control that
> _should_ be used when decreased (mainly z) resolution is acceptable.
> The
>
> lasers can then be turned down and, if desired, decon. can be used
> to help clean up the image. The problem is that many users want a
> "pretty picture" but pretty pictures may not be needed for
> quantification of
> (say) number of mitochondria.  As we say on the Vancouver course, "Every
>
> photon is precious" and you may also increase signal by accepting a
> wider spectral band or using an LP filter.  The key to good experimental
>
> work is to understand what measurement you want and then to pick
> conditions that allow you to get sufficient data with sufficient
> (not too many) time points to answer your question. Do you need a
> full 3D image or will a couple of slices suffice? Use a high NA
> lens. As others have said, consider using widefield with a high QE
> CCD if you really don't need the maximum possible resolution in 3D...
>
> My 2c
>
> Mark Cannell
>
> 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) 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]
> > [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
> > >
> > >
> > >
> >
> >
> >
> >
>
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Dr. Sudipta Maiti
Associate Professor
Dept. of Chemical Sciences
Tata Institute of Fundamental Research
Homi Bhabha Raod, Colaba, Mumbai 400005
Ph. 91-22-2278-2716 / 2539
Fax: 91-22-2280-4610
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