Re: Best Sampling for nucleus quantification in wide-field

Posted by David Baddeley on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Best-Sampling-for-nucleus-quantification-in-wide-field-tp7583395p7583399.html

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Hi Cristophe,
Zdenek has pretty much nailed it, although it has the potential to get a bit (or maybe a lot) more complicated with very high NA lenses. These can have significant apodization of out of focus beam paths which means that you can no longer assume that the sum intensity is constant with depth (we see a 30-40% drop in the total integrated intensity of a bead which  is ~ 2um closer to the objective than the focal point when using our 1.49 NA objective). Testing whether the constant intensity assumption holds for your objective is pretty much as simple as taking a bead stack.  Assuming you're using a reasonable NA, and because yeast nuclei are relatively small, it's unlikely to be much of an issue, but it's one which is worth knowing about. If you do need to work with really high NAs, your best bet would be to take a Nyquist sampled image and then deconvolve it with an experimental PSF before quantifying the intensity.
cheers,David

     On Thursday, 5 February 2015 1:23 PM, Zdenek Svindrych <[hidden email]> wrote:
   

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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 <[hidden email]>
Komu: [hidden email]
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"