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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