I. CONVENTIONAL LIGHT MICROSCOPY remains the core tool in diagnostic surgical pathology. The future of surgical pathology will, however, include electronic modes of image presentation to support diagnoses rendered by viewing the cells or tissue on a computer screen rather than with an optical microscope, since digitized images of glass slides increase the clinically useful information that can be obtained from a pathologic specimen, and permit modes of analysis including electronic consultations, morphometry, and slide storage that go beyond the capabilities of current microscopy-based diagnostic pathology.

II. VIRTUAL MICROSCOPY is the technique whereby entire glass slides or selected areas of slides are scanned and converted to digital data files (also known as virtual slides), which can then be viewed on a computer screen. The characteristics of the displayed image, in terms of resolution and range of magnification, are primarily determined by the optical features of the scanning system and are overall comparable with those of glass slide microscopy. However, in contrast to conventional light microscopy where the magnification, focus, and condenser setting can be adjusted at any time, for digital scanning, the “scanning depth” must be determined prior to image acquisition. The scanning depth includes the region of interest, the scanning power, and the number of horizontal levels to be obtained in the plane of the tissue section (scanning power includes the physical magnification [e.g., 20×] and resolution [e.g., 2048 × 4800 pixels], while the number of horizontal levels [so-called z-stacking] is dependent on the section thickness and the need to be able to focus up and down through the tissue and cells in the final virtual image). Depth of focus is a requirement for interpretation of specimens that rely on a three-dimensional assessment of cellular morphology, for example, cytology specimens (Cancer Cytopathol. 2007;111:203).

Average scanning time for a single horizontal level of a slide (non-z-stacked) is about 5 minutes and generates a data file that is enormous (ranging from about 200 MB to 1.5 GB). Unfortunately, no uniform (open-source) virtual slide file format is currently available, although interfaces are available that can convert image files between the different virtual slide formats.

Each virtual file consists of multiple parts, called file segments, which include an identification tag, a scanned barcode linked to the laboratory information system (LIS), a low-power overview, and the images at the selected power. While image acquisition methods vary (linear, meander, array), currently all available systems produce tiled images, that is, small images that are retrieved and stitched together to form the final image on the computer screen at the selected power. Most interfaces allow electronic zooming (i.e., additional magnification) beyond the original scanned power.

Resolution and functionality of virtual slides have achieved levels that are comparable with conventional light microscopes. Scanning time, on the other hand, is still time-consuming, since scanning time is mainly dependent on computational
speed and optical physics, the latter of which is determined by the magnification of the scanning objective. The actual image acquisition step occurs via a chargecoupled device (CCD), which consists of several hundred thousand individual picture elements (pixels) that transform the optical image into a virtual image; whole slide images are therefore acquired via a single optical lens that moves over the slide. Some scanners shorten scanning time by using a meander rather than a linear scanning pattern to acquire images, but both the meander and the linear methods have inherent physical limitations that become most apparent when acquiring multiple images within each plane of section (i.e., when z-stacking). An emerging technology, so-called lens array microscopy that uses arrays of detectors (miniaturized lenses) to simultaneously capture information from larger areas of the tissue section, may be able to markedly shorten scanning times but still meet the high standards of diagnostic pathology (Hum Pathol. 2004;35:1303).