Re: color calibration of microscopic images

From: Beatrice (pappagallo16_at_libero.it)
Date: 09/13/04 

Date: 13 Sep 2004 07:36:51 -0700

David Littlewood <david@nospam.demon.co.uk> wrote in message news:<nZ9TYhI+nfQBFwqs@dlittlewood.demon.co.uk>...
> >
> >> Do you find a colour shift as you change the exposure time or ISO
> >> setting on the camera?
> >
> >Besides a change in brightness? I would not know for sure. But I can
> >tell you that I have acquired images of the grey patches of the Gretag
> >Macbeth chart, at different exposure times, and I see a nonlinear
> >effect (even before the histogram becomes saturated).
>
> If you mean non-linear changes in luminance, why would this be a
> problem? I did not think you were trying to get an absolute value for
> luminance. I agree, if there is non-linear effect on the chrominance
> this would be a problem.

One of the things I would like to do is to be able to compare images
acquired at different exposure times. At the beginning I thought that
I could, in some way, simply 'rescale' the intesity values of these
images so as if I had acquired them at a single exposure time.

So I measured the color of the Gretag Macbeth grey patches at
different exposure times to determing the scaling factors.
But then it turned out that:
1 - the color of the same grey patch at different exporure times
acquisitions does not increase linearly.
2 - the colors of different grey patches at the same exposure time do
not increase linearly.
These 'crossed' non-linearities are a problem for me as they make (at
least for me) this rescaling more complicated, and I would not know
any more how to do it.

(BTW, then I thought that color calibration would have also solved
this problem, besides having more accurate color reproduction).

>
> Birefringence in the specimen would not be a problem here*; you would
> only using the two polarisers as a light attenuator, and the sample is
> not between them. The retardation of a birefringent sample will only
> show up as a colour effect if it is between the two polarisers. The
> advantage of using crossed polars as an attenuator, compared with using
> ND filters, is that any residual colour from absorption in the
> polarisers should be constant for sample and reference (Macbeth chart or
> whatever) whereas adding ND filters to just one of them risks adding a
> colour cast.
>
> *You might get some anomalies if your specimen was pleochroic.
> Pleochroism is quite different from birefringence, and less common - it
> is having a different absorption spectrum along different axes of the
> sample crystals. However, if your specimens do show pleochroism, this
> would be a whole new ball game - and something that showed it up could
> even be useful.

All right, but with polarizers I would cut away diffused light, which
I think is an important component of the color. Think for example at
the difference between matt and glossy materials.
It is also true that by working in dark field I get rid of specular
reflections, so same problem. But since paint in cross-sections is
more matt than glossy, I think this would be the preferred solution.
What do you think?

> OK, I understand. However, a spectrophotometer would at least allow you
> to calibrate the system to show that your reference samples were
> consistent and your attenuation method was not causing colour shifts.

Good point.
I can add that since I am going to make my own reference samples, I
was thinking to measure their color by using my spectrophotometer and
then calculate RGB coordinates from the spectra. In this case I would
not have any problems of calibration, since for our system I already
have a calibration procedure by comparison with the spectral lines of
a gas discharge lamp.
The spectrophotometer in my lab consists of an Imspector spectrograph
connected to the microscope on one side and to a digital camera to the
other side.
The only thing is that I still have to see how to perform this step
exactly. I think that I would have to take into account the
tristimulus and the characteristics of the light source, don't know
yet exactly how and how easily.

_________________________________________________

Aaron <nghy@comcast.net> wrote in message news:<0ik6k0pnqgkscg9mj27ej0gmnufk802lc6@4ax.com>...
> Hello Beatrice,
>

1)
> Now that I have refelcted a bit on your comments, I favor a
> tristimulus colorimeter which will read out coordinates in the "color
> solid" that represents human color perception. These numerical
> readings plus your own recorded comments will provide the body of
> knowledge that you desire.
>

2)
> The instrumental measurements will make sense over time and will
> perform another function for which the eye/brain is inept, i.e.,
> remebering colors over time.
>

3)
> Konic-Minotla makes a non-contact tristimulus coloimeter Model
> CS-100A which seems to have many characteristics that would be useful
> in your application. I think an instrument of this type could be
> adapted to the output of a microscope and could be used hand held near
> the surfaces of paintings.
>
> See.
>
> http://konicaminolta.com.hk/ph/eng/pdf/catalogue/cs_100a_e.pdf
>
> Good Luck
> Aaron

So, if I understand you, you prefer a tristimulus colorimeter because:
1 - I would have directly color coordinates in a CIELab or similar
space.
2 - since the colorimeter has his own illumination source (if I
understand correctly), I would not have to rely on the microscope
light source whose characteristics are not well known.

About the first point, couldn't I just convert my RGB readings to
CIELab values (which is what I was planning to do after calibration)
without necessarily using a colorimeter?
About the second point, well, this is a good point.
What is not clear to me is whether such colorimeters make
spatially-resolved measurements. If not, then I can't use them.
Please correct me if I am wrong.

Beatrice