For Nyquist as I heard it, you want to sample at a minimum of twice
the spatial frequency of the smallest detail of interest (to you).
Note this is the *minimum* for resolving the detail at all.  I suspect
the three times factor you suggested is to improve on this.  Usually
it is a matter of fiddling with the pixel size you get an 'acceptable'
image by your own standards.  Since your microscope is not an ideal
system rules like this "are more 'guidelines'", to quote Captain Jack
Sparrow. @:-)

Craig


On Wed, Oct 7, 2009 at 9:34 AM, Eric Scarfone <[log in to unmask]> wrote:
> hello list,
> I've try to look into that question but I probably looked in the wrong
> places.
>
> It is widely said that in order to respect the famous Nyquist criteria, one
> should sample signals at 3 times the resolution of the acquisition system.
> Hence if ia calcuate correctly, using a 0.80NA objective to image
> fluorescein at 520nm (which gives a 390nm resolution), one should set the
> zoom of a laser scanning system so that xy pixels have a size of 130nm.
> BUT, what if the structures I am interested in are in the 1 to 5 µm range?
> In that case I do not really need the ful resolving power of my objective
> and using say 300nm pixel size should provide enough sampling to correctly
> image 1000nm objects, or? What do the expert say??
>
> There are  practical aspects to this question.
> - using a high zoom factor exposes living preps to large amount
> of potentially harmfull photons
> - when one scans a limited amount of pixels to increase frame frequency,
> zooming to nyqvist drastically reduces the field that is imaged
> - I am working on pretty old data that I have no possibility whatsoever to
> re-acquire with a higher sampling rate ... ;^)
>
> Thanks for your thougts
>
> Eric
>
>
>
>
>
>
>
> Eric Scarfone, PhD, CNRS,
> Center for Hearing and communication Research
> Department of Clinical Neuroscience
> Karolinska Institutet
>
> Postal Address:
> CFH, M1:02
> Karolinska Hospital,
> SE-171 76 Stockholm, Sweden
>
> Work: +46 (0)8-517 79343,
> Cell: +46 (0)70 888 2352
> Fax: +46 (0)8-301876
>
> email: [log in to unmask]
> http://www.ki.se/cfh/
>
>
>