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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 |
#2
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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 |
#3
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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 |
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