Dear listers,

Could someone let me know if they received my post from Thurs. past 
concerning the optical slice issue. For this topic, I claim no 
special insight that hasn't been repeated a number of times by 
subsequent posts. It is just that more than a few times my 
submissions have been completely ignored. Are others seeing my 
submissions or not? If someone could let me know off list or on I 
would appreciate it. Thanks,

Mario

>Dear Sudipta and All,
>
>(Message from a commercial vendor)
>
>On 08/14/2010 06:36 PM, Sudipta Maiti wrote:
>>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.
>Good point!
>>First, the number of Z-slices required for imaging the bead: more than the
>
>To record a bead or multi-bead image to derive a PSF from it, it should
>be recorded at the Nyquist rate or better. The number of Z-planes should
>be sufficient to fully contain the PSF. In a typical confocal case 30+
>planes should do it. There are a number of caveats though, see for example:
>
>http://www.svi.nl/RecordingBeads
>http://www.svi.nl/PsfFromBeads&highlight=distiller
>
>In short, recording the bead is quite independent of recording the
>biological object, provided that optical conditions are the same. If
>bleaching or time constraints dictate few planes of the object, then
>from the standpoint of deconvolution a slight undersampling in Z is
>acceptable. But not too much!
>
>>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.
>Indeed, the pinhole sizes used in recording the PSF and the data should
>match. The sampling can be adapted and the tails of the PSF can be
>extrapolated though it is better if this is not necessary.
>
>Regarding the tradeoff between pinhole size, microscope type, signal and
>deconvolution, the outcome of that is very much object dependent. As a
>rule of thumb, with small sparse objects you're likely to be better off
>with a WF system + deconvolution, with thick dense objects with a
>confocal system. Increasing the pinhole beyond the Airy disk size gets
>you less Z-resolution and more signal in the absolute sense, but as Guy
>pointed out more (blurry) signal from adjacent areas carries also more
>noise. Deconvolution can repair the resolution loss, again much
>depending on the sparseness of the object. For example, if there are
>strongly labelled horizontal membranes in your image next to other
>interesting but faint features, opening the pinhole is probably not a
>good idea.
>
>Regarding the number of planes in the data to be recorded for
>deconvolution: if possible record enough planes to contain the entire
>object. If that is not feasible, then the more the better.
>In the low noise WF sparse object case even single plane deconvolution
>is possible (this is not 2D deconvolution!), in the confocal case three
>or more Nyquist sampled planes are needed. Of course reliability is not
>as good that of a full-object deconvolution, and there are cases where
>3-plane confocal 'deconvolution' will fail. But as long as the object is
>fairly sparse and there are no large bright objects right outside the
>recorded volume, my colleague Vincent's point was that chances are good.
>The catch is this: because the volume in the data contributing to a
>deconvolved pixel is less in the 3-plane case compared to a full-object
>deconvolution, the signal to noise (SNR) requirements are a bit higher
>than in the full object case. If the choice is between 3 higher SNR
>planes or more lower SNR planes, both cases involving the same amount of
>detected photons, best go for more planes.
>
>We've done simulations on this topic in the past, if anyone is
>interested we could construct a wiki page.
>
>-- Hans
>
>>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
>>>>>
>>>>>
>>>>
>>>>
>>>>
>>>>
>>>
>>>No virus found in this incoming message.
>>>Checked by AVG - www.avg.com
>>>Version: 9.0.851 / Virus Database: 271.1.1/3032 - Release Date: 08/12/10
>>>04:34:00
>>
>>
>>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
>>alternate e-mail: [log in to unmask]
>>url: biophotonics.wetpaint.com
>>
>
>
>--
>-------------------------------------------------------------------
>dr. Hans T.M. van der Voort                           ([log in to unmask])
>Scientific Volume Imaging b.v.,             URL: http://www.svi.nl/


-- 
________________________________________________________________________________
Mario M. Moronne, Ph.D.

[log in to unmask]
[log in to unmask]