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White Paper – November 2015
Digital imaging has been embraced by the research and scientific communities, and concepts such as white balancing, brightness matching and color calibration are often discussed as if they are well-understood and a given. This paper reviews these concepts and identifies where opportunities exist.
Color calibration of digital microscope images of stained biological slides may use a special slide (called a calibration slide) with known color samples that are usually filters. To evaluate quantitatively how well the calibration has been done by a particular system, another set of different known samples is needed, with transmittance spectra that are typical of stained biological material. A slide with such colors comprises what is called a reference slide. In this article, a method is described for selecting the colors for such a slide. A reference slide was created by this prescription and then used to evaluate two embodiments of a color management system for several microscope and illumination combinations. It was found that a set of 20 reference colors spans the gamut of colors produced by a particular kind of stain. Using the reference slide, Datacolor’s ChromaCalTM system performance was quantitatively evaluated. The performance is robust to change of microscope and illumination and does not degrade much when a tungsten light is increased in intensity (hence in correlated color temperature) with only one initial calibration at a single color temperature followed by automatic white balancing of a traditional sort.
Gina Marie Londino, M.S.
Senior Lecturer, Forensic and Investigative Sciences Program, Indiana University Purdue University Indianapolis
Impression Pattern and Trace Evidence Symposium – August 2015
An Innovative Method for Obtaining Consistent Images and Quantification of Histochemically Stained Specimens
Journal of Histochemistry & Cytochemistry 2015, Vol. 63(4) pp 233–243
Michael A. Linden, Gerald J. Sedgewick, and Marna Ericson
Department of Lab Medicine and Pathology, University of Minnesota, Minneapolis, Minnesota (MAL);
Imaging and Analysis, Saint Paul, Minnesota (GJS); and
Department of Dermatology, University of Minnesota, Minneapolis, Minnesota (ME)
Obtaining digital images of color brightfield microscopy is an important aspect of biomedical research and the clinical practice of diagnostic pathology. Although the field of digital
pathology has had tremendous advances in whole-slide imaging systems, little effort has been directed toward standardizing color brightfield digital imaging to maintain image-to-image consistency and tonal linearity. Using a single camera and microscope to obtain digital images of three stains, [Linden, et al.] show that microscope and camera systems inherently produce image-to-image variation. Moreover, [Linden, et al.] demonstrate that post-processing with a widely used raster graphics editor software program does not completely correct for session-to-session inconsistency. [Linden, et al.] introduce a reliable method for creating consistent images with a hardware/software solution (ChromaCal™; Datacolor Inc., NJ) along with its features for creating color standardization, preserving linear tonal levels, providing automated white balancing and setting automated brightness to consistent levels. The resulting image consistency using this method will also streamline mean density and morphometry measurements, as images are easily segmented and single thresholds can be used. [Linden, et al.] suggest that this is a superior method for color brightfield imaging, which can be used for quantification and can be readily incorporated into workflows.
On February 12, 2015, Datacolor hosted a seminar with Dr. Stefan Hamann of Biogen Idec (Cambridge, MA). A recording of the webinar is available here. The audience response was overwhelmingly positive, so Datacolor conducted a follow up interview with Dr. Hamann, delving deeper into some key topics of his webinar.Click Here