A novel consistent photomicrography technique using a reference slide made of neutral density filter

Obtaining consistent photomicrographic images of pathology slides is not always easy because of many different types and settings of the equipment such as the microscopes and digital cameras. In this study, we developed a photomicrography technique that could acquire consistent images of pathology slides. The neutral density (ND) filter was attached to a transparent glass slide as a reference slide, photographed using consistent settings, and acquired images that harbored all of the areas of gray, white, and black. In the same way, the slide was replaced by the actual pathology slide, and photomicrographed. To simulate different light environment, the above photographic session was repeated using two different light intensities and microscopes. A graphic program was used to adjust levels of the reference slide images and this leveling, or calibration, was saved as a file for each. This file for leveling process was applied to actual subsequent photomicrographic images. The same sites of noncalibrated and calibrated images of the pathology slide were calculated into CIELAB or CIE L*a* coordinates. Then, the color differences (ΔE*ab) were calculated. As results, in the study using a 50% transmittance ND filter, two original different images were made nearly identical to the unaided eye, especially in two-point (white and gray) and three-point (black, white, and gray) leveling. In comparison of different light intensities, the ΔE*ab of the selected area was 0.9 in two-point leveling. Between different microscopes, 10.7 of ΔE*ab was the smallest value in three-point leveling. This method would be helpful for acquiring consistent photomicrographic images of pathology slides.