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The diagonal in z will be much 'straighter' (due to the fact that the voxels are elongated in z rather than being square), making the factor much closer to 1 (probably something like 1.1) so it can safely be ignored. When talking about slightly oversampling, 2.3 is already doing this - strict Nyquist is a factor of 2. It's also worth noting that you should probably use the theoretical resolution values (ie ~180x450 for a 1.4 NA objective @500nm and a pinhole of 0.7 AU) and not the observed PSF width, as these reflect the bandwidth of the system. I this tend to reccommend a blanket 70x70x200nm pixel size when using a high NA objective on fixed cells. In live cells, or other delicate samples you need to exercise a little more discretion - the artefacts introduced by slight undersampling are likely to be outweighed by other considerations.
My 2c,
David
------------------------------
On Thu, Apr 12, 2012 3:44 AM NZST Vasseur Monique wrote:
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>Hi John,
>
>Indirectly, do you suggest the same for Z sampling if we are interested in 3D measurements? Thanks
>
>Monique Vasseur
>
>-----Message d'origine-----
>De : Confocal Microscopy List [mailto:[log in to unmask]] De la part de Lemasters, John J.
>Envoyé : 11 avril 2012 09:34
>À : [log in to unmask]
>Objet : Re: Nyquist and Image size
>
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>Please remember that pixel spacing on the diagonal is 1.4 that in the horizontal and vertical directions. Accordingly to meet the Nyquist criterion for the diagonal, pixel size should be 2.3 x 1.4 = 3.2. Also, the Nyquist criterion is an arbitrary threshold, and image quality will improve somewhat with sampling greater that proposed by Nyquist. Considering diagonal sampling, I suggest using a pixel size that is one fourth of the resolving limit for the most critical work.
>
>John
>
>--
>John J. Lemasters, MD, PhD
>Professor and GlaxoSmithKline Distinguished Endowed Chair Director, Center for Cell Death, Injury & Regeneration Departments of Pharmaceutical & Biomedical Sciences and Biochemistry & Molecular Biology Medical University of South Carolina
>DD504 Drug Discovery Building
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>
>-----Original Message-----
>From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of John Oreopoulos
>Sent: Wednesday, April 11, 2012 8:29 AM
>To: [log in to unmask]
>Subject: Re: Nyquist and Image size
>
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>Renato,
>
>Whether you have 256x256, 512x512 or 2048x2048, the "optimum" Nyquist sampling rate (ie: pixel dimensions) does not change since your objective lens did not change. The quoted pixel size at 2Kx2K you mentioned (22.5 nm x 22.5 nm) means you are oversampling the image (and not gaining anything). Your image may look smoother but it contains no more information than the 512x512 image with 90x90 nm pixel sizes. Presumably the scan speed is the same between 512x512 and 2Kx2K.
>
>You should decrease the galvometric mirror scan zoom setting to get back to an effective pixel size of 90x90 nm with 2Kx2K pixels in your image. Effectively, you will be imaging (and properly sampling) a larger field of view then. I'm not familiar with the Leica laser scanning confocals so I'm not sure if it will allow you to do this. On other systems, like the Olympus FV300 for example, you can set your image pixel dimensions (256x256, 512x512, etc.) and your scan zoom independently.
>
>Just out of curiosity, why image 2K x 2K when you can't easily display that on a standard computer screen or present it in a published paper without downsizing? I rarely departed from 512x512 in my laser scanning days, except when I wanted to see a larger field of view.
>
>Cheers,
>
>
>John Oreopoulos
>Research Assistant
>Spectral Applied Research
>Richmond Hill, Ontario
>Canada
>www.spectral.ca
>
>
>On 2012-04-11, at 7:22 AM, Renato Mortara wrote:
>
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>>
>> Dear all,
>>
>> Having attended the first Pawley course in Vancouver I feel highly
>> embarassed to ask this, but I would really appreciate a clarification:
>>
>> When estimating the highest zoom users should apply to their sample in
>> order to accommodate for the Nyquist theorem, I estimated the optimum
>> pixel size value by dividing the lateral resolution (eg: 0.2 microns)
>> by 2.3 so that the value is approxiametely 90 nm.
>>
>> The doubt: if the image size is increased from 512x512 (having
>> adjusted the zoom to the pixel size of 90nm) to 2Kx2K, the resulting
>> pixel size (displayed by the system - Leica) the pixel size decreases
>> 4 fold, to 22.5 nm. Since the resolution obviously did not change but
>> only the image size, what happens to Nyquist and the optimum pixel size at 2Kx2K ?
>>
>> Many thanks !
>>
>> Renato
>>
>> Renato A. Mortara
>> Parasitology Division
>> UNIFESP - Escola Paulista de Medicina
>> Rua Botucatu, 862, 6th floor
>> São Paulo, SP
>> 04023-062
>> Brazil
>> Phone: 55 11 5579-8306
>> Fax: 55 11 5571-1095
>> email: [log in to unmask]
>> home page: www.ecb.epm.br/~ramortara
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