Hi sinead
On 23 Jul 2009, at 06:00, IMAGEJ automatic digest system <
[hidden email]
> wrote:
>
> Thank you very much for your help, I am afraid I know very little
> about t=
> he principles of image=20
> analysis and processing,
>
>
If you look at some teaching material I just made on quantitative
digital
Image analysis you might get some tips
https://info.med.tu-dresden.de/MTZimaging/images/9/95/Quant_imag_and_image_processing0709.pdfThe rgb weighting is because our eyes our most sensetive to green,
less to red and least to blue.
However your ccd camera or other digital detector has a different
response to different colours.
It is worth checking the detector technical spec to see the quantum
efficiency as a function of light wavelength
To measure fluorescence in similar samples prepared on different or
even the same day you must internal fluorescence intensity controls.
Perhaps fluorescent beads.
It is hard to compare the intensity of two different dyes absolutely.
Relative measurements are easier.
Don't assume the fluorescence intensity in a sample is linearly
related ( directly proportional) to the dye concentration. That is
probably not strictly true because detectors are often non
Linear in their response and fluorescence intensity depends on the
chemical/physical environment of the dye which is different in
different parts of the sample. Concentrations should be measured
biochemically to confirm imaging results.
Don't saturate the detector and clip off the real values of your
brightest objects. Use a range indicator or Hilo look up table to set
exposure and gain and offset of your detector properly
Quantification of saturated images under estimates the real intensity
Cheers
Dan
Daniel J. White Ph.D.
Max Planck Institute - CBG
Light Microscopy Facility
and Image Processing Facility.
Dresden, Germany.
http://www.bioimagexd.nethttp://www.chalkie.org.ukMobile +49 15114966933
Office +49 351 210 2627
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