normalization of fluorescence intensity quantification

[jiami Guo]

Hi,

I am interested to show the fluorescence intensity difference of a lineage marker staining between stem cell and differentiating stem cell. I am thinking maybe I can just use nuclear staining as normalization to control for the focal plane differences and photobleaching. I want to measure area and intensity of each single cell and normalize each cell against their nuclear staining intensity. Can anybody tell me what's the best way to do it? Or is there better way to do quantify my staining? Any suggestion will be good! Thank you!

Jiami Guo

[Daniel James White]

Hi Jiami Guo,

Watch out! You are in danger of making basic mistakes here...


On Nov 20, 2010, at 6:00 AM, IMAGEJ automatic digest system wrote:

> ------------------------------
>
> Date:    Fri, 19 Nov 2010 09:11:31 -0500
> From:    jiami Guo <[hidden email]>
> Subject: normalization of fluorescence intensity quantification
>
> Hi,
>
> I am interested to show the fluorescence intensity difference of a lineage marker staining between stem cell and differentiating stem cell.

it is very hard to compare the fluorescence intensity between different samples, as you cant be sure the staining was exactly the same.
You need internal fluorescence brightness controls, like fluorescent beads mounted next to the sample.

> I am thinking maybe I can just use nuclear staining as normalization to control for the focal plane differences and photobleaching.

be very very careful here.... there are many reasons why this is not a robust , reproducible approach....
its not a good idea to try to measure the relative intensity between 2 different dyes/channels.

the onlt robust measurement is a relative intnsity between adjacent areas of the same sample looking at the same fluorescent dye/channel...
and even then, a non uniform illiumination can catch you out - esp on a confocal with a lower mag lens.

> I want to measure area and intensity of each single cell and normalize each cell against their nuclear staining intensity.

you can do it, if you have fluorescence instyernsity controls IN the samples... ie fluorescent beds or similar,
that are always the same brighness, so you can use as an internal control, and normalise signals to that.

without controls, your measurement will be unreliable.

> Can anybody tell me what's the best way to do it? Or is there better way to do quantify my staining? Any suggestion will be good! Thank you!
>

you need to read and absorb
http://www.sh.lsuhsc.edu/oor/rcf/Microscopy/Basic%20Microscopy%20Info/Pawley.pdf

then have a hard thing about how to design your experiment in a robust way.

this is not trivial... so feel free to ask more specific questions... help is out there!


cheers

Dan



> Jiami Guo
>

Dr. Daniel James White BSc. (Hons.) PhD
Senior Microscopist / Image Processing and Analysis
Light Microscopy Facility
Max Planck Institute of Molecular Cell Biology and Genetics
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Germany

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