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Just to add a bit to John's absolutely correct explanation.
It's basically a question of interpretation. You set up Nyquist imaging at 512x512 and then selected 2048x2048, expecting to get the same resolution but a 4 times bigger area. Perfectly reasonable. But the Leica software assumed you now wanted to get 2048x2048 pixels within the same chosen field of view. Also a perfectly reasonable interpretation. One might, in a perfect world, expect the software to ask you which interpretation you want, but if it doesn't it's pretty easy to fix.
Guy
Optical Imaging Techniques in Cell Biology
by Guy Cox CRC Press / Taylor & Francis
http://www.guycox.com/optical.htm
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-----Original Message-----
From: Confocal Microscopy List [mailto:[log in to unmask]] On Behalf Of John Oreopoulos
Sent: Wednesday, 11 April 2012 10:29 PM
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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> To join, leave or search the confocal microscopy listserv, go to:
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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
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> 04023-062
> Brazil
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> home page: www.ecb.epm.br/~ramortara
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