EH 6_2015

SPECI AL:DIGITALPATHO LOGY EUROPEAN HOSPITAL  Vol 24 Issue 6/15 12 DIGITAL PATHOLOGY Digital microscopy will upgrade w Digital pathology: a new diagnostic technologyHistopathologists play key roles in diagnosing disease entities and determining biomarkers related to the prognosis and response to specific therapy of malignant tumours Histopathology is still firmly based on cell and tissue morphology supple- mented with in situ molecular infor- mation and these together can be studied using an optical microscope. Digital microscopy creates a dig- ital representation of the whole microscopic slides at decent quality, which can be dynamically viewed, navigated and magnified via a mouse and computer monitor, and shared though computer networks without spatial and temporal limita- tions. Digital slides can be integrated into the hospital information system (HIS) and accessed through intra- or internet for teach- ing, primary d i a g n o s i s , teleconsul- tation and q u a l i t y a s s u r - ance. Discrete pixels of calibrated quali- ties allow automated image analysis and signal quantification for draw- ing unbiased conclusions in diag- nostic and research applications. Therefore, utilisation of the full power of computer technology to access multiple functions and the internet grants digital microscopy great potential to upgrade the effi- ciency of pathology workflow and pathologists. By resolving critical issues, including standardisation of data formats, secure and fast inter- net communication and medico- legal aspects, digital microscopy is expected to play a revolutionary role in future histopathology. Digital slides created by slide scanners Digital microscopy creates large digital files representing all crucial details of stained tissue sections with decent resolution and high colour fidelity achieved using auto- mated focusing and white balance. Digital slides are made up of giant arrays of rectangular pixels organ- ised along x-y coordinates, each of which is characterised by size, col- our and intensity values. Produced either by area or line scanning, digital slides are built up as pyramids of microscopic image series where low power views are generated by compressing the origi- nal sharp and optimally lit images (Fig. 1A). Scanning through several focal levels within the usual 3-8μm sample thickness offers access to the z dimension used for emulat- ing fine focusing of the optical microscope. Fig. 1 A) The \image series by compression allows view- ing of the digital slide at arbitrary magnifications (e.g. x15 and x10). Scanning at different focal levels within the sample thickness (~3- 8μm) offers access to fine details in the z-dimension. Using x10 lens offers high field of view (FOV) size and focal depth, while requiring small storage space. A x20 objective allows double the optical resolution than that of x10, but at the expense of revealing smaller FOV and focal depth, while increasing the storage need. x40 objective does not offer significant improvement in optical resolution compared to x20 (NA=0.9 vs.0.8) despite needing large storage space and long scanning time. Unique features of digital slides Seeing slides on a monitor, with easy access to a computer’s multi- functionality, is far more ergonom- ic than peering though an ocular lens of an optical microscope. Even pathologists with high affection for conventional microscopy respect digital microscopy benefits if they practise enough. The computer-gen- erated image pyramid format of digital slides allows in-focus naviga- tion through continuously chang- ing magnifications, without chang- ing objectives, or realigning the focus or lighting conditions. Digital magnifications beyond that used for scanning still reveal fine micro- scopic details hidden in the original magnification. Slides can be tilted arbitrary for proper orientation and preview images of the whole slide are avail- able simultaneously on the monitor where navigation history of high power analysis can also be traced (Fig. 3). Fig. 3 A) Digital slide viewer interfaces utilise the whole comput- er monitor where preview images and navigation history (left side) of high power analysis can also be traced. Calibrated pixels allow straight measurements of object dis- tance, perimeter or area highlighted by permanent annotations. B) The monitor can be shared for several digital slides for comparative stud- ies, as shown by the same area of serial slides stained for H&E, the proliferation marker Ki67 (brown; middle) and the gap junction con- nexin-43 (red immunofluorescence) combined with the Ki67 protein (green; right), respectively in oral epithelial hyperplasia. Permanent annotations and text put on digital slides, straight meas- urements of object distance, perim- eter or area and prompt still-image Fig. 1 A) The image pyramid generated from optimised x20 magnification image series by compression allows viewing of the digital slide at arbitrary magnifications (e.g. x15 and x10). See details wihin the article. Fig. 2 Schematic representation of area scanning (A, B) and line scanning (C, D) techniques used by available slide scanners. A) Classical area scanners collect large series of images at x-y dimensions through a microscope objective with a CCD camera, either in bright-field or fluorescence mode. B) The area scanner combining an 80-element lenslet array with complementary metal oxide semiconductor (CMOS) sensor can cover large section areas at once. C) Typical line scanners can collect image strips from the continuously moving slides through an objective using a linear array light sensor, which is, however, not sensitive enough for fluorescence signals. D) Combination of 64 or more of linear array sensors permits TDI (time delay and integration) scanning, where consecutive sensors cumulate the signals making TDI appropriate also for fluorescence scanning 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. Bela Molnar MD DSc is CEO of 3DHISTECH Ltd, which has developed high-performance hardware and soft- ware products for digital pathology since 1996. A medical graduate from Semmeweis University, Budapest, and the Hungarian Academy of Sciences, he gained qualifications as an internist and gastroenterologist, followed by a post doctoral 2-year fellowship with Boehringer Ingelheim GmbH. Scientific/industrial cooperation arose with Roche Diagnostics, Epigenomics Inc (Seattle/Berlin), and Carl Zeiss MicroImaging. Today, Molnar’s main research areas include biomarkers of colorectal cancer development, molecular biology applications, virtual microscopy, and quantitative image analysis, and his pub- lications, memberships of scientific bodies and professional awards are numerous.

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