http://confocal-microscopy-list.275.s1.nabble.com/Optical-slice-thickness-and-number-for-PSF-and-deconvolution-tp5415740p5424453.html
Quite so, you need to know the PSF that exists in the _sample_. So if
water (shudders) that is what you need to do to your beads too. That
> 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 [
[hidden email]] On Behalf Of Sudipta Maiti [
[hidden email]]
> Sent: Saturday, August 14, 2010 12:36 PM
> To:
[hidden email]
> 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:
[hidden email]]
>> On Behalf Of Mark Cannell
>> Sent: Friday, 13 August 2010 8:52 AM
>> To:
[hidden email]
>> 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
>>>
[hidden email]
>>>
[hidden email]
>>> 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
>>>>
[hidden email] <mailto:
[hidden email]>
>>>> 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:
[hidden email]
> url: biophotonics.wetpaint.com