Digital Pathology

14 Fig. 4 Some FISH signals of HER-2 gene (red) and CEP17 (green) of less than a micron remain hidden from single focus photography (A), but can be revealed when multiple focus layers are scanned and then projected (B; extended focus). Please note that several green signals that are missing from A are clearly seen on B. C) Accumulation of all red and green signals gained from merging consecutive optical layers allows reliable analysis. D) FISH (fluorescence in situ hybridisation) signals revealed in a cell can be intensity-amplified in 3-D (E) for better assessment. F) Cell nuclei can be automatically sorted into groups in a gallery according to their FISH pattern and re-localised into their tissue environment Unique features of digital slides Seeing slides on a monitor, with easy ac- cess to a computer’s multi-functionality, is far more ergonomic than peering though an ocular lens of an optical microscope. Even pathologists with high affection for conven- tional microscopy respect digital microsco- py benefits if they practise enough. The computer-generated image pyramid format of digital slides allows in-focus navigation through continuously changing magnifica- tions, without changing objectives, or re- aligning the focus or lighting conditions. Digital magnifications beyond that used for scanning still reveal fine microscopic details hidden in the original magnification. Slides can be tilted arbitrary for proper orientation and preview images of the whole slide are available simultaneously on the monitor where navigation history of high power analysis can also be traced (Fig. 3 A). Digital slide viewer interfaces utilise the whole computer monitor where preview images and navigation history (left side) of high power analysis can also be tra- ced. Calibrated pixels allow straight measure- ments of object distance, perimeter or area highlighted by permanent annotations. The monitor can be shared for several digital slides for comparative studies, as shown by the same area of serial slides stained for H&E, the proliferation marker Ki67 (brown; middle) and the gap junction connexin-43 (red immunofluorescence) combined with the Ki67 protein (green; right), respectively in oral epithelial hyperplasia. (Fig. 3B) Permanent annotations and text put on di- gital slides, straight measurements of ob- ject distance, perimeter or area and prompt still-image archiving at publication quality all support the pathology workflow. Sever- al digital slides can be opened side-by-side on the monitor for comparative analysis of serial slides of a sample stained for diffe- rent biomarkers. Even samples of immu- nohistochemistry and FISH (fluorescence in situ hybridisation) can be opened, linked and navigated alongside, which normally need consecutive steps or even separate microscopes. Pixels making up the images have calibrated dimension, colour and intensity, features that can be used for colour separation-ba- sed automated quantification and measure- ments of image-objects. Furthermore, pattern recognition of mor- phological and functional units within the tissue, such as glands, or hyperplastic or abnormally arranged epithelial nests, can be automatically made based on shape, size and texture identification. Serial digital slides can also be assembled into a 3-D structure for reconstructing tissue architecture, e.g. to study tumour invasion or re-orient colorectal biopsies. Digital data, including whole digital slides with annota- tions and measurements, can be integrated into digital databases and shared though intra- or internet with unlimited partners, even simultaneously. The freedom to ac- cess digital slide archives for re-review, and the logistics of slide storage and sorting are

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