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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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