Re: Best Sampling for nucleus quantification in wide-field

Posted by Steffen Dietzel on
URL: http://confocal-microscopy-list.275.s1.nabble.com/Best-Sampling-for-nucleus-quantification-in-wide-field-tp7583395p7583400.html

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Am 05.02.2015 um 20:59 schrieb Christophe Machu:
>  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 ?

Sure. Provided you do a few controls: Uniformity of illumination,
linearity of the detector, uniformity of labeling intensity (e.g.
antibody concentration and penetration), absence of quenching and
fluorescence saturation......

Seriously? if you have a 10x increase, no problem. If you have a 10%
increase, you need to work a lot on controls and provide huge numbers
(with good statistics) to convince a reviewer who knows the subject.
Have a look at Jim Pawley's 39 steps. Although written for confocal
quantitative fluorescence, much of it also applies to widefield. One link:
http://labs.pbrc.edu/cellbiology/documents/39steps.pdf

Good luck.

Steffen

>
> --
> ------------------------------------------------------------
> Steffen Dietzel, PD Dr. rer. nat
> Ludwig-Maximilians-Universität München
> Walter-Brendel-Zentrum für experimentelle Medizin (WBex)
> Head of light microscopy
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> Marchioninistr. 27
> D-81377 München
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