http://confocal-microscopy-list.275.s1.nabble.com/Best-Sampling-for-nucleus-quantification-in-wide-field-tp7583395p7583398.html
I never thought of that. I will test myself your solution ASAP. Can I then conclude from what you say that the integrated intensity I’ll be measuring is representative of the whole amount of tagged protein in the nucleus ?
I am trying to correlate fluorescence intensity with amount of protein.
And to reply to Peter, yes I would like to extract some morphological and localisation features from the images, so flow cytometry won’t bring me enough information on that aspect.
Thanks guys for your rapid input I really like this community and might come again with more questions in the near future.
> On 05 Feb 2015, at 19:22, Zdenek Svindrych <
[hidden email]> wrote:
>
> *****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
http://www.imgur.com and include the link in your posting.
> *****
>
> 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
>
> "*****
> To join, leave or search the confocal microscopy listserv, go to:
>
http://lists.umn.edu/cgi-bin/wa?A0=confocalmicroscopy> Post images on
http://www.imgur.com and include the link in your posting.
> *****
>
> 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"