Fluorescence Intensity

[Sam Angel]

Hello.  I was wondering whether someone could help me please. I am running four different experiments which I hope will correlate at the end of the day. The basis is the same, I am looking at the effect of growing brain cells (1 normal brain cell line, 1 brain tumour (high passage), and 2 brain tumours (low passage)) in 3 different sera (human, serum free and fetal calf). For two of the experiments (flow cytometry and immunocytochemistry), the cells are then exposed to 8 different antibodies. With Flow Cytometry, the geometric mean fluorescence intensity shows that there is a trend in both cell type and the sera in which the cells are grown. What I would like now is to do some stats with the ICC. I have today discovered ImageJ, but cannot find appropriate instructions. Unfortunately, my micrographs haven't been taken in a standard fashion, (number of cells in the field of view, exposure, brightness, contrast, etc); whereas my Flow data was all captured with one standard set of the instrumentation. Will it be possible to standardise my images in order to compare them? How best would I get some data? If at all possible?

Your help is very much appreciated.
Kind regards,
Sam

[Gabriel Landini]

On Friday 16 January 2009 15:17:03 samantha murrayI wrote:

> Hello.  I was wondering whether someone could help me please. I am running
> four different experiments which I hope will correlate at the end of the
> day. The basis is the same, I am looking at the effect of growing brain
> cells (1 normal brain cell line, 1 brain tumour (high passage), and 2 brain
> tumours (low passage)) in 3 different sera (human, serum free and fetal
> calf). For two of the experiments (flow cytometry and immunocytochemistry),
> the cells are then exposed to 8 different antibodies. With Flow Cytometry,
> the geometric mean fluorescence intensity shows that there is a trend in
> both cell type and the sera in which the cells are grown. What I would like
> now is to do some stats with the ICC. I have today discovered ImageJ, but
> cannot find appropriate instructions. Unfortunately, my micrographs haven't
> been taken in a standard fashion, (number of cells in the field of view,
> exposure, brightness, contrast, etc); whereas my Flow data was all captured
> with one standard set of the instrumentation. Will it be possible to
> standardise my images in order to compare them?

... [show rest of quote]

No.

G.

[Sam Angel]

Sam Angel wrote

Hello.  I was wondering whether someone could help me please. I am looking at the effect of growing brain cells in 3 different sera (human, serum free and fetal calf). I would like to see the effect of the sera on 8 different antibodies by flow cytometry and immunocytochemistry. The flow cytometry analysis is complete, I would now like to compare the ICC. I have now standardised my ICC micrographs and was wondering how I can use ImageJ to quantify the fluorescence intensity? I probably wouldn't need to subtract the background as I have appropriate background ICCs. I have around 200 micrographs, so some sort of automation/plugin would be good.

Your help is very much appreciated.
Kind regards,
Sam