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Hello, everyone,

Just like to emphasize one thing that others already mentioned:   The
photobleaching rate is proportional to the SQUARE of the zoom-factor.
Therefore, increasing zoom unnecessarily (beyong Nyquist sampling rate)
will bleaching the sample much faster. Therefore, you may even get a
worse (bleached) picture.

Xuejun

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
>
>
>Assoc. Prof. Guy Cox,                 ooOOOOOOoo
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>

--
Xuejun Sun, Ph.D.
Dept. Exp. Oncology
Cross Cancer Institute
11560 University ave.
Edmonton Alberta T6G 1Z2
Canada

Phone:  (780) 432-8898 (office)
        (780) 432-8468 (lab.)
Fax:    (780) 432-8425




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