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Theoretically it is totally right to assume that one can either increase the sampling frequency or the field of view if the number of pixels are increased.
Practically one cannot increase the field of view in a scanning system without getting nasty side effects like e.g. uneven illumination. To my understanding this is the reason why vendors tend to go for option one if you increase the number of pixels.
Cheers Arne
---------------------------------------------------------------
Arne Seitz
Head of Bioimaging and Optics Platform (PT-BIOP)
Ecole Polytechnique Fédérale de Lausanne (EPFL)
Faculty of Life Sciences
Station 15, AI 0241
CH-1015 Lausanne
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Fax: +41 21 693 9585
http://biop.epfl.ch/
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> -----Original Message-----
> From: Confocal Microscopy List
> [mailto:[log in to unmask]] On Behalf Of Guy Cox
> Sent: mercredi 11 avril 2012 15:06
> To: [log in to unmask]
> Subject: Re: Nyquist and Image size
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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
> ______________________________________________
> Guy Cox, MA, DPhil(Oxon), Honorary Associate, 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 John
> Oreopoulos
> Sent: Wednesday, 11 April 2012 10:29 PM
> To: [log in to unmask]
> Subject: Re: Nyquist and Image size
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
> http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy
> *****
>
> 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:
>
> > *****
> > 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
> > 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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