Re: Nyquist and Image size

Posted by Craig Brideau on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Deconvolution-of-Transmission-Images-tp7393699p7456788.html

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I've always thought of Zoom as my field-of-view control.  Many microscope
systems let you set a sub-region to scan, and depending on the software
this can be either zoom or crop.  Some software lets you select whether it
acts as either or when you readjust the size of the sub-region selection
box.

Craig


On Wed, Apr 11, 2012 at 7:05 AM, Guy Cox <[hidden email]> wrote:

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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
> ______________________________________________
> Guy Cox, MA, DPhil(Oxon), Honorary Associate,
> Australian Centre for Microscopy & Microanalysis,
> Madsen Building F09, University of Sydney, NSW 2006
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> -----Original Message-----
> From: Confocal Microscopy List [mailto:[hidden email]]
> On Behalf Of John Oreopoulos
> Sent: Wednesday, 11 April 2012 10:29 PM
> To: [hidden email]
> 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: [phone]
> > Fax:     [phone]
> > email: [hidden email]
> > home page: www.ecb.epm.br/~ramortara
>