color calibration of microscopic images

Hi all,

I would like to calibrate the color of images acquired with a digital
camera attached to a microscope. My samples consist of thin section
taken from paintings. I have collected a lot of information about
color calibration and I set up a procedure, but I am having some
problems.
Since I do not have much practical experience, at some point it
becomes difficult for me to establish the quality of what I obtain.
Maybe someone with more experience could help me in this sense?

My first problem is the color standard target.
As far as I know there are no color calibration targets on the market
for usage under the microscope. Perhaps someone has knowledge of any
such target? What I am currently using is a Gretag Macbeth ColorChart,
which under the microscope does not look homogeneous at all. Also, at
the exposure times that I use for acquiring images of my samples, many
of the color patches of the Gretag chart are overexposed.
I acquired images of the Gretag chart patches individually and I
calculated the average (RGB) color for each patch. By comparing these
colors with the real color values I see that the colors that I measure
are much more vivid than the real ones. I wonder how much of these
color differences are due to the instrumental setup characteristics or
to the fact that I am viewing the chart under high magnification.

Still, I wanted to try how the calibration works under these
conditions.
I have a Matlab routine which can perform a sequence of a linear and a
non-linear (third-order polynome) color calibration. The routine seems
to perform quite well, by looking at the acquired chart after color
correction. Corrected colors are much more similar to the real ones,
maybe a bit greysh; it improves by excluding from the calibration the
patches of lighter colors, that tend to saturate.

But when I apply the obtained calibration to the sample images, the
resulting image has a very dull appearance.
By looking at the histograms of the images before and after
calibration, it's clear that there is a loss of contrast, as the
histograms become narrower.

I might somewhat expect this result, as the calibration is obtained by
comparing an image of the calibration chart with very vivid and bright
colors (that is what I acquire) with one with darker colors (the real
ones). My problem is that I have no idea of how a correctly
color-calibrated image should look like, therefore how much my results
are close (or far) from the correct result. Unfortunately nothing of
what I have read do any mention of the problems I have found.
I am wondering whether the source of the problem resides in the
calibration routine or in the type of color standards that I am using.

Can anyone help me having a better understanding? Should I maybe try a
different type of color standard (what about the IT8 charts on
photographic paper?)?

Thank you!

Beatrice

In article , Beatrice
writes
Hi all,

I would like to calibrate the color of images acquired with a digital
camera attached to a microscope. My samples consist of thin section
taken from paintings. I have collected a lot of information about
color calibration and I set up a procedure, but I am having some
problems.
Since I do not have much practical experience, at some point it
becomes difficult for me to establish the quality of what I obtain.
Maybe someone with more experience could help me in this sense?

My first problem is the color standard target.
As far as I know there are no color calibration targets on the market
for usage under the microscope. Perhaps someone has knowledge of any
such target? What I am currently using is a Gretag Macbeth ColorChart,
which under the microscope does not look homogeneous at all. Also, at
the exposure times that I use for acquiring images of my samples, many
of the color patches of the Gretag chart are overexposed.
I acquired images of the Gretag chart patches individually and I
calculated the average (RGB) color for each patch. By comparing these
colors with the real color values I see that the colors that I measure
are much more vivid than the real ones. I wonder how much of these
color differences are due to the instrumental setup characteristics or
to the fact that I am viewing the chart under high magnification.

Still, I wanted to try how the calibration works under these
conditions.
I have a Matlab routine which can perform a sequence of a linear and a
non-linear (third-order polynome) color calibration. The routine seems
to perform quite well, by looking at the acquired chart after color
correction. Corrected colors are much more similar to the real ones,
maybe a bit greysh; it improves by excluding from the calibration the
patches of lighter colors, that tend to saturate.

But when I apply the obtained calibration to the sample images, the
resulting image has a very dull appearance.
By looking at the histograms of the images before and after
calibration, it's clear that there is a loss of contrast, as the
histograms become narrower.

I might somewhat expect this result, as the calibration is obtained by
comparing an image of the calibration chart with very vivid and bright
colors (that is what I acquire) with one with darker colors (the real
ones). My problem is that I have no idea of how a correctly
color-calibrated image should look like, therefore how much my results
are close (or far) from the correct result. Unfortunately nothing of
what I have read do any mention of the problems I have found.
I am wondering whether the source of the problem resides in the
calibration routine or in the type of color standards that I am using.

Can anyone help me having a better understanding? Should I maybe try a
different type of color standard (what about the IT8 charts on
photographic paper?)?

Thank you!

Beatrice

Hi Beatrice,

Wow, you don't believe in any easy life, do you.

First thought: you will find vastly more expertise on microscopy, film
and digital, on sci.techniques.microscopy and on the Yahoo Microscopes
mailing list. The people there are universally helpful and mostly very
knowledgeable indeed.

Second, this is not a subject I have ever seen discussed before. Part of
the reason for this is that there are just too many variables to contend
with. FWIW - and I don't claim any great expertise - here are some
thoughts, though they are more questions than answers. (I think you will
get much more informed responses in the above two groups if you can
include these details.)

1 How big are your colour chips, and what kind of microscope are you
using? If they are of reasonable size, do you need to use a compound
microscope? If you can use a simple microscope or photomacroscope this
might simplify your problems.

2 Are these chips opaque or transparent - IOW, are you using incident
light (epi-illumination) or transmitted light? Is the light source
built-in or separate? You might find it easier to calibrate a separate
source (see 3).

3 I would imagine that the first requirement for such work is a
consistent and calibrated light source. This would normally (for a
compound microscope) be an incandescent bulb of some type (tungsten, or
tungsten/halogen usually) plus quite a bit of optics (mirrors, condenser
etc). Changes in colour temperature due to slight changes in voltage can
be large, as can changes as the bulb ages, and even the other optics
could have some effect.

4 Many pigments and dyes show odd effects - pleochroism (different
absorption spectrum in different axes) and metamerism (different colours
in different light) would be the most common.

5 Is there any reason for needing to use the microscope approach? If
your chips are large enough, would it be useful to examine them directly
with a colour densitometer? Also, a spectrophotometer may give you much
more unambiguous information about the absorption (or reflection, as the
case may be) properties of the material.

6 Macbeth do provide standard patches for calibrating their
densitometers, but I am not sure what range of colours they offer.

7 A bit more off the wall - have you thought of calibration using a
standard quartz wedge and a Michel-Levy interferometric colour chart?

8 Most important of all, some more specific details of what you are
trying to achieve will help the experts in the other places give you a
better answer.

Regards (and good luck),

David
--
David Littlewood

In article , Beatrice
writes
Hi all,

I would like to calibrate the color of images acquired with a digital
camera attached to a microscope. My samples consist of thin section
taken from paintings. I have collected a lot of information about
color calibration and I set up a procedure, but I am having some
problems.
Since I do not have much practical experience, at some point it
becomes difficult for me to establish the quality of what I obtain.
Maybe someone with more experience could help me in this sense?

My first problem is the color standard target.
As far as I know there are no color calibration targets on the market
for usage under the microscope. Perhaps someone has knowledge of any
such target? What I am currently using is a Gretag Macbeth ColorChart,
which under the microscope does not look homogeneous at all. Also, at
the exposure times that I use for acquiring images of my samples, many
of the color patches of the Gretag chart are overexposed.
I acquired images of the Gretag chart patches individually and I
calculated the average (RGB) color for each patch. By comparing these
colors with the real color values I see that the colors that I measure
are much more vivid than the real ones. I wonder how much of these
color differences are due to the instrumental setup characteristics or
to the fact that I am viewing the chart under high magnification.

Still, I wanted to try how the calibration works under these
conditions.
I have a Matlab routine which can perform a sequence of a linear and a
non-linear (third-order polynome) color calibration. The routine seems
to perform quite well, by looking at the acquired chart after color
correction. Corrected colors are much more similar to the real ones,
maybe a bit greysh; it improves by excluding from the calibration the
patches of lighter colors, that tend to saturate.

But when I apply the obtained calibration to the sample images, the
resulting image has a very dull appearance.
By looking at the histograms of the images before and after
calibration, it's clear that there is a loss of contrast, as the
histograms become narrower.

I might somewhat expect this result, as the calibration is obtained by
comparing an image of the calibration chart with very vivid and bright
colors (that is what I acquire) with one with darker colors (the real
ones). My problem is that I have no idea of how a correctly
color-calibrated image should look like, therefore how much my results
are close (or far) from the correct result. Unfortunately nothing of
what I have read do any mention of the problems I have found.
I am wondering whether the source of the problem resides in the
calibration routine or in the type of color standards that I am using.

Can anyone help me having a better understanding? Should I maybe try a
different type of color standard (what about the IT8 charts on
photographic paper?)?

Thank you!

Beatrice

Hi Beatrice,

Wow, you don't believe in any easy life, do you.

First thought: you will find vastly more expertise on microscopy, film
and digital, on sci.techniques.microscopy and on the Yahoo Microscopes
mailing list. The people there are universally helpful and mostly very
knowledgeable indeed.

Second, this is not a subject I have ever seen discussed before. Part of
the reason for this is that there are just too many variables to contend
with. FWIW - and I don't claim any great expertise - here are some
thoughts, though they are more questions than answers. (I think you will
get much more informed responses in the above two groups if you can
include these details.)

1 How big are your colour chips, and what kind of microscope are you
using? If they are of reasonable size, do you need to use a compound
microscope? If you can use a simple microscope or photomacroscope this
might simplify your problems.

2 Are these chips opaque or transparent - IOW, are you using incident
light (epi-illumination) or transmitted light? Is the light source
built-in or separate? You might find it easier to calibrate a separate
source (see 3).

3 I would imagine that the first requirement for such work is a
consistent and calibrated light source. This would normally (for a
compound microscope) be an incandescent bulb of some type (tungsten, or
tungsten/halogen usually) plus quite a bit of optics (mirrors, condenser
etc). Changes in colour temperature due to slight changes in voltage can
be large, as can changes as the bulb ages, and even the other optics
could have some effect.

4 Many pigments and dyes show odd effects - pleochroism (different
absorption spectrum in different axes) and metamerism (different colours
in different light) would be the most common.

5 Is there any reason for needing to use the microscope approach? If
your chips are large enough, would it be useful to examine them directly
with a colour densitometer? Also, a spectrophotometer may give you much
more unambiguous information about the absorption (or reflection, as the
case may be) properties of the material.

6 Macbeth do provide standard patches for calibrating their
densitometers, but I am not sure what range of colours they offer.

7 A bit more off the wall - have you thought of calibration using a
standard quartz wedge and a Michel-Levy interferometric colour chart?

8 Most important of all, some more specific details of what you are
trying to achieve will help the experts in the other places give you a
better answer.

Regards (and good luck),

David
--
David Littlewood