Slide scanners are the highest level of abstraction for digital microscopy. They have both a hardware and software components. We distinguish five levels, from lowest to highest: slide handling, slide scanning, optics, detection, and, finally, acquisition software. These five levels are depicted in Fig. 2.2.

The first slide scanner was designed by James Bacus in 1994, during a period of rapid Internet expansion worldwide. The corresponding BLISS system, which is now recognized as having been the first virtual microscopy system ever developed, was designed to generate virtual microscope slides. Meanwhile, a WebSlide Server, Browser, and ActiveX Viewer were developed to allow for viewing virtual microscope slides over the Internet. Over the next several years, the Bacus group developed and patented the methods and apparati to perform automated assays of biological specimens, immunoploidy analysis, measurements of tissue thickness, and tests of neoplasm progression, as well as devices to allow for the remote control of microscopes, the creation of virtual microscopy slides, the magnification of specimen images, and the Internet, intranet, and local viewing of such slides [1–19]. Moreover, Bacus Laboratories not only created the first virtual slide system, they also created the first market for it. They did this by framing their system as an educational tool. Their plan was to ultimately replace standard microscopes with virtual microscopy in medical education. They achieved this by combining their virtual microscopy system with a collection of educational “slides,” for which institutions could lease access licenses annually. Because of its successful business model, Bacus Laboratories was purchased by Olympus America Inc. in 2006.

In terms of features, the capacities of slide scanners today vary widely. For example, some can do bright-field images only, some can do fluorescence images only, and some can do both. The price of a model generally correlates with its slide-loading capacity, which can range from one to 400 slides per batch. Slides can be handled as a single slide/stage, as standalone autoloaders (“hotels”), as slide trays, or as slide magazines. The type of slide can vary as well: While most scanners today still handle basic 1” × 3” slides only, others—like Aperio and Huron Technologies—also support 2” × 3” and even larger (whole mount) slides. The more variability that is allowed for physical slide media, the harder it is to batch process large numbers of slides.

Two approaches exist to scanning a slide: tile scanning (Bacus patents) [20] and line scanning (Aperio patents) [21]. In both cases, the resulting images (tiles or strips) are fitted together into a single large image (i.e., the WSI). With a tile scanner, the slide is scanned as a series of rectangular tiles. For each tile, the highest physical magnification desired is used (e.g., 40× or 20×). The tiles are then stacked into a WSI, like bricks forming a wall. This is done either concurrently with or after the scanning process, via the acquisition software. Conversely, with line scanning, after magnification, strips are combined side by side into a single image. Proponents of the latter approach claim that it generates fewer seams and, hence, fewer optical aberrations (Fig. 2.3).

One particular problem related to scanning is focusing. A pathologist looking through a microscope automatically adjusts the focus depending upon the area of the slide he or she is looking at, the thickness of the specimen, the type of glass slide used, etc. With a scanner, this process must be automated. With both tile and line scanners, it is possible to autofocus each field after moving the stage, but this can be very time-consuming, especially with tile scanners. A better approach is to focus on every nth field being scanned. This is both faster and simpler; but the placement of focus points lacks context, and it is still possible to waste time on larger areas that, by chance, do not require refocusing. A focus map is another solution. With this approach, focus points are distributed over the tissue forming a surface. Focus is only recalculated for intervening tissue. The number of selected focus points can be controlled via the scanner software. A trade-off is usually made between more focus points (less speed) and greater accuracy. The settings can be tissue dependent, and a technician can maintain a preset list of “profiles” that can be referred to, depending upon the type of specimen that needs to be scanned.

Z-stacking is becoming increasingly commonplace, but this poses its own unique challenges to file format organization (see later in this chapter). The new frontier is now spectral imaging.