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Guy et al--

In the older pre-Radiance BioRad systems, the zoom was performed using a
scan card that allowed (if I remember correctly) 2^12 positions for each
galvanometer.  Thus, for a 512 x 512 scan, you could get "true" zooms of
1, 2, 4 or 8x.  However, zooms at other values were approximated by
double-scanning some lines--i.e. they were not entirely accurate and
introduced some artifacts.

Is this still the case for the Radiance systems?  If so, I wonder if
that might be the issue underlying the (perhaps garbled) warning that
she received.

Regards--

Martin Wessendorf

Guy Cox wrote:

> Search the CONFOCAL archive at
> http://listserv.acsu.buffalo.edu/cgi-bin/wa?S1=confocal
>
> Gary wrote:
>
>>This question has just been posed and I need a correct answer.
>>
>>I know that zooming on our Radiance 2000 when using a 63x oil
>>objective anymore than 2 to 3 times is a "waste."  The precise
>>definition of "waste" is what I'm looking for.  I have been told that
>>this will yield a digital artifact, due to artificial "filling," as a
>>result of decreased resolution.
>
>
>
> You have been given somewhat inaccurate information.  'Zooming'
> on a confocal is just scanning a smaller area, so there is no
> digital filling involved.  The key question you need to ask is
> how many pixels you need to actually get the full resolution
> your lens can give you - with this lens 200nm or better.
>
> The Nyquist criterion says that you need a minimum of 2.3 pixels
> within the minimum resolved distance to be able to capture that
> resolution in a digital image.  At zoom 3 your Radiance has a pixel
> size of 120nm so it is clear that you will NOT use the full resolution
> of your lens at zoom 3 or below.  At zoom 4 you are getting fairly
> close to Nyquist (pixel size 90nm).
>
> I believe that a _small_  amount of oversampling is a good thing
> (your lens should do a bit better than 200nm and/or you may want
> to smooth the image a bit to reduce noise, etc. etc) so I'd go to
> a maximum of 3 pixels per minimum resolved distance.  This isn't
> enough to make the image look fuzzy - in fact for visual presentation
> it looks pretty well perfect.  This takes you somewhere between
> zoom 5 (pixel size 70nm) and zoom 6 (pixel size 60nm).
>
> Anything above zoom 6 will start to look fuzzy since you are in
> 'empty magnification' - there just isn't any fine detail to see
> and your eye doesn't like that.  Also you will bleach your sample
> more - to no purpose.  So make zoom 6 the limit with this lens.
>
> All these figures are based on 512 x 512 image - obviously this
> will be different for different image resolutions.  But the Bio-Rad
> always shows you the actual pixel size so it's easy to see if you
> are in the right area.
>
>                                                         Guy
>
>
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--
Martin Wessendorf, Ph.D.                   office: (612) 626-0145
Assoc Prof, Dept Neuroscience                 lab: (612) 624-2991
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