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Hi Christophe,
congratulations for choosing widefield!

You don't need any z-sampling. A single image contains the fluorescence from
the whole nucleus, as long as the nucleus is at least approximately in focus
and the signal does not overlap with the fluorescence from other nuclei.

You may try it yourself - take a z-stack of a single nucleus in your FOV, 
the total fluorescence should be constant over a fairly wide range of 
defocus (of course you need to include all the fluorescence, including the 
out-of-focus blur).

Depending on the intensity, size and density of your nuclei, you might 
benefit from lower NA objective (you'll get lower intensity, but also less 
blur).
Best, zdenek

Btw, not a silly question at all!

-- 
Zdenek Svindrych, Ph.D.
W.M. Keck Center for Cellular Imaging (PLSB 003)
University of Virginia, Charlottesville, USA
http://www.kcci.virginia.edu/workshop/index.php



---------- Původní zpráva ----------
Od: Christophe Machu <[log in to unmask]>
Komu: [log in to unmask]
Datum: 5. 2. 2015 11:57:18
Předmět: Best Sampling for nucleus quantification in wide-field

"*****
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Hello everyone !!

I have a very silly question. In our lab we would like to be able quantify
the whole signal intensity within a yeast nucleus. I know how to measure it
easily but I have doubts the Z sampling I am using. Should I sample using a
Nyquist criterion (but counting several time the same molecule in the
nucleus) or should I assume the theoretical Z resolution of my microscope
(knowing my objective specifics) ?

I would personally vote for the second option but I'd be glad to be
corrected if I am wrong.

Thanks a lot in advance for our inputs.

Best, Christophe"