Yes, your post came through. I didn't comment because at the time I was
embroiled in an off-list debate with Mark Cannell on the same topic.
(It seems our differences were largely terminology - he regards setting
the pinhole at 1 Airy unit to be opening it up whereas I regard that as
keeping it closed!)
As to your post, I just cannot accept that deconvolution is 'essential'.
It may give you a prettier picture, and at best it may well show you
things that you couldn't see before, which has obvious value. But the
downside is that you end up with an image that is non-quantifiable, and
to me that is a big sacrifice. I use deconvolution, and have even
written simple deconvolution software, but I cannot accept that it is
essential.
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 Mario
Sent: Tuesday, 17 August 2010 1:27 AM
To: [log in to unmask]
Subject: Re: Optical slice thickness and number for PSF and
deconvolution
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.
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