EMS Catalog #10-1010
About Color Calibration
1. Why should I consider color calibration as an important part of my digital microscopy work?
In brightfield imaging, it is widely observed that the colors from the same specimens look differently between microscopes, cameras and monitors. Sometimes the variation occurs between imaging sessions even though the same equipment is used. Without consistency, how confident can you be with the images and the evaluation?
The ChromaCal™ System is a first-of-its-kind color calibration system for optical microscopy. ChromaCal™ enables researchers, scientists and technicians to establish and preserve the color integrity of their digital transmitted, brightfield microscopy images. By standardizing color reproduction, ChromaCal™ delivers a consistent, reliable basis for evaluation, communication, documentation and publication.
ChromaCal™ also reduces your imaging efforts and workflow. Through an easy-to-use batch function, ChromaCal™ quickly processes specimen images in a single step that are:
- Color standardized
All of thisis achieved in a controlled and documented process so the integrity of your research is secure.
Bottom line: With ChromaCal™, you achieve better image quality, improved comparability, less effort, documented changes, and batch processing. So eliminate your time-consuming efforts in image-editing software (e.g. Photoshop) and eliminate the possibility of unintentionally compromising your research data.
2. Do I have to calibrate my monitor(s)?
Using the entire ChromaCal™ System (i.e. combining calibrated monitors with image calibration) delivers color integrity to the entire imaging workflow.
However, if you choose not to use ChromaCal™ Image Calibration software to color-calibrate your images, it is still highly recommended that you calibrate all monitors that are used to display your specimen images. This allows you (and your colleagues and collaborators) to achieve a consistent viewing and communication experience.
Monitor calibration is included within the ChromaCal™ Color Calibration System (Catalog #10-1010). However, the ChromaCal™ Monitor Calibration Set (Catalog #10-3010) can also be purchased separately. For example, certain institutions may not perform brightfield microscopy but still understand the need for reliable and consistent color rendering on their displays. The ChromaCal™ Monitor Set can address this need and function independent of the ChromaCal™ Image Calibration software.
If your monitor is either uncalibrated or was not calibrated using a ChromaCal™ sensor, a notification message will appear on the main screen of your ChromaCal™ Image Calibration software.
3. Is white balancing sufficient to standardize the color of my images?
Although white balancing is a necessary step in calibrating color, it is not sufficient. ChromaCal™ Image Calibration software utilizes a standardized color matrix to establish and apply a color profile to digital color images. With these capabilities, color can be standardized within your lab, among your facilities, and between collaborators.
Note that, as part of the color-calibration feature, ChromaCal™ also performs automatic white balancing as well as matching brightness of your specimen images. As a result, you can avoid the time and effort often spent in image-editing software (e.g. Photoshop) to white balance and adjust brightness.
About the ChromaCal™ Product Configurations and Licenses
1. What is included in the ChromaCal™ Color Calibration System case? Is anything else required to use the ChromaCal™ System?
The ChromaCal™ Color Calibration System case (Catalog #10-1010) consists of the following:
- ChromaCal™ Image Calibration software
- ChromaCal™ Monitor Calibration software
- ChromaCal™ Sensor for use with the ChromaCal™ Monitor Calibration software
To utilize the ChromaCal™ Image software, you will also need to purchase a ChromaCal™ Color Calibration Slide for Brightfield Microscopy (Catalog #10-2010). To promote color standardization within your facility, ChromaCal™ Image Calibration software accommodates the use of multiple ChromaCal™ slides. So, locating a slide at each microscope or with each technician or microscopist is recommended to encourage use and provide easy access.
2. Can I use ChromaCal™ software on more than one workstation?
Your original purchase of the ChromaCal™ Monitor Calibration software allows you to install this software on three (3) workstations, so you may want to calibrate the monitor at your microscope, at your analysis workstation and at your desk.
Your original purchase of ChromaCal™ Image Calibration software allows you to install this software on one (1) workstation, typically where you process your captured digital images.
In either case, if you wish to install any ChromaCal™ software on additional workstations, additional activations can be purchased directly through the software, whenever a new workstation installation exceeds your available activations. When ordering additional activations, you will need to provide the license code provided with your original software purchase.
3. Can I use the ChromaCal™ slide on multiple microscopes?
The ChromaCal™ Color Calibration Slide is highly portable and can be used on any brightfield microscope. To promote color standardization within your facility, ChromaCal™ Image Calibration software accommodates the use of multiple ChromaCal™ slides. So, locating a slide at each microscope or with each technician or microscopist is recommended to encourage use and provide easy access.
4. Why does the ChromaCal™ Monitor Calibration software allow 3 workstation installations but the ChromaCal™ Image Calibration software only allows 1?
Typical imaging systems include three monitors that should be calibrated: one at the microscope, one at an image analysis workstation, and one at the researcher's desk. As a result, three monitor calibration activations are included. On the other hand, ChromaCal™ Image Calibration software is typically installed on the image analysis workstation, and therefore only one workstation activation is included.
If you wish to install any ChromaCal™ software on additional workstations, additional activations can be purchased directly through the software, whenever a new workstation installation exceeds your available activations. When ordering additional activations, you will need to provide the license code provided with your original software purchase.
5. What is Viewer mode and the ChromaCal™ Viewer?
ChromaCal™ Image Calibration software (starting with version 1.7) offers a special Viewer mode. This ChromaCal™ Viewer mode can be installed on an unlimited number of workstations and is provided free of charge with your purchase of the ChromaCal™ System. The ChromaCal™ Viewer offers unlimited use of two specific features:
- ChromaCal™ Linearity Check
Ability to open a ChromaCal™ Color Calibration slide image that is generated from your imaging system, and then receive a diagnostic evaluation of the linearity of your system. Using this feature, you can regularly check the linearity of each of your imaging systems, and this check can be conveniently performed at the workstations attached to those systems... all without using a Licensed activation of the ChromaCal™ software. Linearity is critical, so we've made it simple and readily available.
Please note: A ChromaCal™ Color Calibration slide is required for the linearity check.
- ChromaCal™ Viewer
Ability to open and view any images that have been color-calibrated by ChromaCal™, whether created by you or shared with you by another ChromaCal™ user. Using this feature, you can share the power of the ChromaCal™ Viewer (and ChromaCal™-calibrated images) with all your colleagues and collaborators... simply provide them with the ChromaCal™ Image software and they can activate the Viewer on their own workstations without needing additional Licensed activations. This is truly a gift that you can keep on giving!
Please note: ChromaCal™ images are saved in industry-standard formats (TIFF and JPEG), so these images can be readily viewed with any standard image-viewing software. ChromaCal™ doesn't create proprietary formats that restrict sharing. The ChromaCal™ Viewer is specifically designed to display ChromaCal™-calibrated images and should be your viewer of choice for your ChromaCal™ images.
About Microscope and Camera Requirements
1. Are there any microscope requirements or restrictions?
Your microscope should be configured for transmitted light illumination of the specimen. ChromaCal™ Image Calibration System works independently from the microscope hardware and can be used with any manufacturer's microscope system. You should, however, follow good microscopy practices.
2. Does ChromaCal™ work with any camera?
A scientific grade camera with manual exposure setting and white balancing is recommended for image acquisition. Many consumer-grade and some older cameras do not have adequate dynamic range. ChromaCal™ requires raw (original, unprocessed) images saved in TIFF or JPEG format.
About the ChromaCal™ Color Calibration Slide
1. Why is there no cover slip on the ChromaCal™ Color Calibration Slide?
The ChromaCal™ slide uses narrow wavelength filters that retain their spectral properties when left uncovered. The ChromaCal™ calibration algorithm has been optimized to work without a cover slip on the calibration slide, even though specimen slides may typically include a cover slip.
2. Could you describe the configuration of the color matrix on the ChromaCal™ slide?
In the center of the ChromaCal™ Color Calibration Slide is a chip composed of two color matrices each consisting of 20 color circles in a 4(h) x 5(w) configuration.
The larger matrix consists of 30µm color circles and generally accommodates objective magnifications of 4X up to 40X, depending on any intermediate magnifiers or reducers in the camera coupler.
The smaller matrix is configured identical to the large matrix except the color circles are only 9µm in diameter, and generally accommodate objective magnifications from 40X to 100X.
The color circles behave in a similar fashion as bandpass filters in fluorescence microscopy. The colors used in the matrices were selected to cover the visible spectrum for optical microscopy, spanning from 400nm-700nm.
3. What is the expected useful life of the ChromaCal™ slide?
The ChromaCal™ Color Calibration Slide should be handled with care and stored in its slide holder when not in use. Under normal storage conditions and use, the slide is warranted for six (6) months. Use of the slide beyond one year from purchase is not recommended. Additional slides can be purchased separately (Catalog #10-2010). See the warranty for further details.
As with glass slides, the ChromaCal™ slide (and the chip) may collect dust, debris and other contaminants. To reduce the contamination of your slide, be sure to store it in its protective case when not in use. Use a microscope puffer or dry air to gently "blow off" any dust that sits on the calibration chip. DO NOT physically clean (do not rub, wipe, sweep, etc.) the chip area itself. DO NOT use solvents of any kind on the calibration slide (water is considered a solvent).
4. Why are two color matrices provided on the ChromaCal™ slide?
For your convenience, the ChromaCal™ slide offers two color matrices each consisting of the same 20 colors in a 4 (h) x 5(w) configuration. Since the calibration slide is used after your specimen images are captured, the flexibility of two color matrices is simply to allow you to keep your microscope's objective at its most recent setting and just select the color matrix that fits within the applicable viewing area.
5. Do I need to take an image of the ChromaCal™ slide with each objective?
If all of your microscope objectives are of the same optical performance level (i.e. achromat, fluorite/fluor/fluar, semi-apochromat, apochromat, etc.), then you need only acquire one image of the ChromaCal™ slide to use across your specimen images taken during that same imaging session. However, because optical performance level is related to color correction, and if you use more than one performance level of microscope objective during a given microscopy session, it is highly recommended that you acquire an image of the ChromaCal™ slide for each objective type used during that session.
6. How do I clean my ChromaCal™ slide?
It is best to keep your ChromaCal™ slide in its case when not in use; however, dust may still gather on the slide during normal use. Use a microscope puffer/blower or dry air to blow off the dust.
DO NOT rub or wipe the chip itself, even with a cotton swab or lens tissue. Although resilient, the thin film used to create the color patches is subject to mechanical scratching which would result in poor calibration results. DO NOT use solvents or liquids of any kind to clean the calibration slide (water is considered a solvent).
7. Can I use oil or water immersion objectives with the ChromaCal™ slide?
No. Although suitable for objective magnifications up to 100X, removal of immersion oil or water and subsequent cleaning of the ChromaCal™ chip may damage the integrity of the color circles. Please use only "dry" or "air" objectives with the ChromaCal™ slide.
8. Why is the ChromaCal™ slide image so dark when looking through the microscope?
The color circles act like bandpass filters allowing transmittance of only a narrow spectrum of light. Additionally, some colors do not transmit as visibly as others. As long as the whitest patch is visible (and not over-exposed), the ChromaCal™ slide image will provide the data necessary for color calibration of specimen images. It is important to capture the image of the ChromaCal™ slide using the same acquisition settings as the specimen images captured during the same microscopy session.
9. I see what appear to be small imperfection(s) on some of the colored circles on the ChromaCal™ slide. What could these be and will the calibration be affected?
As with glass slides, the ChromaCal™ slide (and the center chip) may collect dust, debris and other contaminants during use. To reduce the contamination of your slide, be sure to store it in its protective case when not in use. Use a microscope puffer or dry air to gently "blow off" any dust that sits on the calibration chip. DO NOT physically clean (do not rub, wipe, sweep, etc.) the chip area itself. DO NOT use solvents of any kind on the calibration slide (water is considered a solvent).
In addition, there may be minor imperfections on the color circles which are a normal by-product of our manufacturing process. As part of our quality assurance testing, each slide and color matrix are inspected. Any minor imperfections will not have an impact on the calibration.
About Images and the Image Files
1. What image format do I need to save in for my specimen images and the ChromaCal™ slide image?
ChromaCal™ Image Calibration software works with raw (original, unprocessed) images saved in either TIFF or JPEG format. TIFF is generally recommended as it preserves the maximum amount of image data.
The use of 8-bits for the bit depth is recommended, although ChromaCal™ Image Calibration software will accommodate images with greater bit depth. Since 8-bit is generally required for output devices, the calibrated images created by ChromaCal™ will be saved as 8-bit files to match what will be seen in print and on monitors. (Note: your original specimen images are preserved, untouched by ChromaCal™).
2. Does ChromaCal™ Image Calibration software alter my original specimen image files?
No, your original specimen image file is preserved. All calibration adjustments are made to a duplicate copy of your original image file. This copy is saved using the naming convention "[original file name]" plus "chromacal-calibrated" added to the end of the file name.
3. Does ChromaCal™ Image Calibration software maintain the linearity of the specimen images?
The algorithms used by ChromaCal™ Image Calibration software apply and maintain a gamma equal to 1. So, if the original image is linear, the calibrated image will also be linear.
However, if the original image is non-linear or had a non-linear enhancement applied, the calibrated image will possess the same non-linearity as in the original image.
About Microscope and Camera Settings
1. Do I need to "structure" the light going to the sample (Koehler illumination)?
Koehler illumination is recommended, since images without Koehler illumination result in reduced resolution. Additionally, the image of the ChromaCal™ Color Calibration Slide may "bloom" without Koehler illumination (i.e. the bright white circle may affect neighboring colors) and therefore the specimen image colors may not be most accurately color calibrated.
2. Is there a light source that does not affect color when turning the light up and down?
Both xenon and LED light sources can be dimmed or brightened without any effect on the color of the image, as long as white balancing took place at the beginning of a microscope session. White balancing is necessary because all light sources affect the color that is either viewed through the eyepiece or captured by the camera.
3. Can I use the camera on the automatic exposure setting?
We do not recommend that you use automatic exposure for your camera, except at the beginning of the session to automatically find your exposure. Automatic exposure could result in widely varied exposures. For that reason, it is better to set exposure manually and use the same exposure setting for the duration of the imaging session.
4. Can I use the camera on the automatic white balance setting?
We recommend that you use manual white balance on your camera or acquisition software, and do not white balance again once you have set your illumination intensity and camera exposure.
5. In the camera software, how should I set the image enhancement settings, such as contrast and color enhancement?
Some camera software allow you to enhance the live-view image before saving it. Such image enhancements are often done by gamma adjustment or certain color correction routines that are intended to make the image more appearing; however, such enhancements change the linearity of the image. We highly recommend you to disable such image enhancement functionality to ensure the optimal color calibration by ChromaCal™.
About ChromaCal™ Image Calibration Software Features
1. Will ChromaCal™ Image Calibration software alter the annotations on my images?
Any annotations made to the uncalibrated image (i.e. scale bar, date stamp, arrows, text, etc.) would be subject to color change with the calibration process.
2. Why do the specimen images appear darker in ChromaCal™ Image Calibration software?
Many software packages auto-brighten images for display purposes. ChromaCal™ Image Calibration software shows the image without auto-brightening, as it appears along an x-axis with the full dynamic range (versus auto-brightening that scales the dynamic range so the image always looks better on screen). The ChromaCal™ method and resultant view is consistent with the tones in the image, and better represents how the image will appear in print, in Acrobat, and likely in Microsoft products as well.
3. How can ChromaCal™ standardize color when it allows the user to modify contrast? Doesn't contrast affect color?
Contrast adjustments do impact color, but based on the individual preferences of microscopists, ChromaCal™ Image Calibration software incorporates flexibility for a controlled range of contrast adjustment. However, this contrast slider will maintain the linear relationship of the color components (red, green, blue) as contrast is adjusted. And, the default contrast level within the software is set to 0 which will result in the highest level of color consistency.
ChromaCal™ Image Calibration software does not employ a gamma-based contrast adjustment since such a method alters the relationship between RGB in a non-linear manner.
About Monitors and ChromaCal™ Monitor Calibration
1. What kind of monitor is best?
ChromaCal™ Monitor Calibration software will work with virtually any monitor. Nonetheless, we are often asked for a recommendation on what kind of monitor would provide the optimal viewing experience. We recommend the use of monitors that have wide viewing angles. This is important because colors and tones change when you look off-angle at inexpensive monitors. This is particularly obvious on laptop displays, but can also plague many inexpensive desktop monitors.
Most inexpensive monitors use a technology called "Twisted Nematic (TN)" with narrow viewing angles. However, monitors that include "In-Plane Switching (IPS)" technology offer wider viewing angles and therefore are recommended.
IPS monitors are not significantly more expensive than TN monitors. These IPS monitors are made by the major manufacturers, but check the specifications before purchasing as only specific models (e.g., the LP2475w from HP) are IPS-based. There are also high-end IPS models from companies such as Eizo and Barco.