Quite so, you need to know the PSF that exists in the _sample_. So if
you open the pinhole to keep the cell alive or even use an oil lens in
water (shudders) that is what you need to do to your beads too. That
said, if the object is noise reduction and not resolution, you may not
be interested in 3D at all, and in that case you case use just 2D
deconvolution to help control noise.
Hope this helps.
Mark
> ar 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
> alternate e-mail: [log in to unmask]
> url: biophotonics.wetpaint.com
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