Posted by
Gabriel Landini on
URL: http://imagej.273.s1.nabble.com/Flat-field-correction-of-fluorescent-images-tp5000936p5000983.html
> I dark current noise image was for high gain and exposure (g8 clk20). I am
> calibrating the fluorescence intensity for all the settings. I did a
> histogram and there are like 13 pixels of 4092 value. I tried it for lower
> gain value and there is still pixels at 4092 just fewer ( g7 clk10 - 5, g7
> clk3 - 1). Should I remove the hot pixels by denoise before using the
> image?
> The camera is MEGA-10 from stanford photonics.
Shouldn't the minimum of the darkfield be very near 0 instead of 191? I guess
that you have too much gain.
Another source of problems is that the camera gets hot as you go along the day
and the bias increases. So the darkfield should be captured before you are
intending to snap the images.
Yes, you will get erroneous image values for these 13 bright pixels as they
output the maximum value, not a biased one (maybe they do, but it eventually
saturates due to long exposure or they might get binned at 4096 by the 8 bit
histogram?).
If you know exactly where they are, you can ignore them, or replace their
value with the average of their neighbours. Many consumer cameras do this,
with a median filter. In the wiki there is a macro to shows how to do this
only for the hot pixels.
I think that for darkfield images (unlike brightfield), you do not compute the
transmittance (i.e. dividing by the background and rescaling), but just
subtract the backgound image. You should be able to capture without a specimen
or estimated by several methods (curve fitting to background points or
morphological filtering). Please correct me if I am wrong.
Cheers
Gabriel
--
ImageJ mailing list:
http://imagej.nih.gov/ij/list.html