A High‐Resolution Multifocal‐Plane Virtual Microscope for Teaching Histology

Abstract

Virtual microscopy (using a computer to view digitally‐scanned microscope slides) is widely employed in teaching histology labs. The increase in the memory capacity of computer servers now makes it possible for virtual microscopy to display virtual slides whose resolution equals that obtained using “real” microscope slides and microscopes with high numerical aperture (NA) oil‐immersion objectives. Because high NA objectives have a very narrow depth‐of‐field, the ability to focus up‐and‐down through the specimen becomes very useful when examining high‐resolution images. We describe here a process for creating multifocal‐plane virtual‐microscope slides whose resolution approaches the theoretical diffraction limit for a light microscope (~250nm) and the creation of a virtual microscope, intended for use in histology teaching labs, that allows the user to focus up‐and‐down through these high‐resolution, multifocal‐plane virtual slides.Our first step in building a high‐resolution, multifocal‐plane virtual microscopy system was to scan histology slides using a Zeiss AxioImager.M2 microscope equipped with a 63x NA 1.4 PlanApo oil‐immersion objective. The final digital magnification of these specimens is 14.4 pixels/μm, giving a theoretical “digital resolution” for these specimens of: 2 pixels ÷ 14.4 pixels/μm = ~140nm. The “digital resolution” is decreased somewhat by the RGB camera’s Bayer filter, but the overall resolution obtained for virtual‐slides scanned by this system is close to the theoretical “optical resolution” of ~250nm.The second step in building our virtual microscopy system was to convert the scanned digital image into a format suitable for our microscope viewer; we chose to use jpeg images in an image‐tile architecture similar to that used by Google Maps. Each focal plane of a virtual‐slide is exported from the Zeiss (*.czi) format as a separate bigTIFF file, and the resulting TIFF file is then converted into a Google‐Maps‐compatible image pyramid using the libvips image processing library. Thus, a multifocal plane virtual slide is converted into a series of separate, but aligned, image pyramids.The final step was to create a virtual microscope that is capable of displaying our multifocal plane digital specimens. HTML/javascript programming was used to create a client‐side viewer that runs within the user’s internet browser. This viewer allows the user to zoom‐in/out to view the specimen at varying magnifications, and to focus up‐and‐down through the specimen. Most of the computing needed to display the specimen is done on the client‐side by the viewer, with the viewer only calling on the server for information about the slide and for image tiles. To provide a responsive interface, the viewer maintains a buffer of images from the focal planes and zoom‐levels adjacent to the image currently being viewed.One of the keys to understanding the microscopic anatomy of an organ is being able to visualize the 3‐dimensional relationships of the cells and tissues comprising the organ. The virtual microscopy system described here clearly demonstrates the utility of being able to focus through a histological specimen at high magnification when attempting to visualize these 3‐dimensional relationships.