Virtual Slide: Real Microscopy – Redefined

Microscopy & Imaging

dotSlideVirtual Slide System

2 CONTENTS

SLIDE INTO THE FUTURE …… todayMicroscopy and image analysis are generally considered to be two separate processes, the formerfeeding into the latter. In some fields though, such as pathology, the two are synonymous withpractitioners analysing preparations directly through the eyepiece. This is due the ‘look and feel’only a real slide-mounted sample and microscope can offer. The dotSlide system from Olympusbrings microscopy and imaging closer together. The result is an advanced and extremely versatile‘virtual’ slide which is an exact copy of the real specimen. Stored electronically, it is accessiblearound the world instantly and simultaneously – the future of imaging is here.

dotSlide in pathology and research 4–9Virtual slide technology enables the information from one glass slide to be sharedacross the globe for teleconsultation, supraregional collaborations, archiving ofslide materials, and teaching. The dotSlide features include: remote access tovirtual slides with the functionality of a microscope, but without the instrumentitself; the ability to see and analyse macro overviews of slides not possible usingtraditional microscopes.

dotSlide – functions and system 10–15All of the dotSlide system components are designed to interact seamlessly, pro-ducing a fully automated, high-speed scanning system with excellent flexibilityand simple operation. These ensure that users get the best results for each typeof specimen with the minimum of effort.

Blood smear, 100x oil. Courtesy of Dr Victor Gisiger,Pathologie et Biologie cellulaire, Université de Montréal,Montreal, Canada.

dotSlide IN PATHOLOGY ANDRESEARCHExceed the limitsVirtual slide microscopy offers both pathologists and researchers the unique potential to acquirecomplete slides at high magnification and resolution, to enable diagnosis, analysis and archiving ofsamples for discussion both remotely or in online conferences. Virtual slide technology enablesusers to exceed the limits of conventional digital microscopy and telepathology in both the size andthe resolution of image files that can be discussed, as well as in time and location of discussionwith colleagues.

CHAPTER I4 dotSlide IN PATHOLOGY AND RESEARCH

dotSlide IN PATHOLOGYWith the increasing demand for networking in hospitals and collaborationscomes the need for the correct tools. The new dotSlide concept from Olympushas introduced a unique high-speed, whole-slide scanning system, producingdigital slide images at high-end microscope quality for pathology. Now, the usercan work more flexibly and efficiently to evaluate slides, diagnose and consultcolleagues for second opinions, at separate times and in different locations.

Virtual slide – real diagnosisTaking an exact copy of an entire slide using the dotSlide system increases the flexibility of case analysis. This unique ability is due to the careful combination ofprecision optical components, high-quality scanning stage and camera with ad-vanced application software.

The whole pictureThe careful integration of all the components creates a highly flexible system thatenables the user to acquire digital slides for diagnosis quickly and effortlessly. Theacquisition process starts with generating an overview image of the entire slide. Itincludes the automatic detection of the specimen to avoid time-consuming scanningof areas where there is no sample on the slide. As a second step, the high-magnifica-tion scan is automatically performed, over the whole slide or over smaller, predefinedareas of interest using the preselected objective. During the acquisition, thousands of individual images are simultaneously compressed without any loss in quality andstitched together to create a seamless, magnified version of the slide. As a result, thisis carried out within minutes, even using high-magnification objectives.

Worldwide accessThe virtual slide image is stored with all the relevant information in such a way that it enables instant, controlled, access from anywhere in the world. This makes it anexcellent tool for remote diagnosis (telediagnostics), second-opinion consultations,group discussions and intra-operative diagnosis of frozen sections. The system pro-vides each user with an easy-to-use on-screen control interface, allowing free navi-gation within the sample. Moreover, the users can control multiple virtual slides and/orother image file formats simultaneously for easy comparison. In this way, they canaccess virtual slides from wherever they are and therefore do not need to be in theiroffice or even their home country to screen a slide.

Meeting roomMore importantly, as well as giving individual access to the virtual slides, the systemalso enables collaboration around the globe via the access-controlled conferencingtool. This allows users to be assigned different roles such as moderator, speaker orclient. Therefore, second opinions can be requested or difficult case discussions canhappen immediately and in real time, with the added benefit of annotation, highlightingand minute taking – a truly revolutionary tool ensuring a new level of efficiency.

Biopsy

Sample preparation

Sample digitalisation

Evaluation and teleconsultation

6 dotSlide IN PATHOLOGY AND RESEARCH

Teaching toolTeaching new students to analyse pathology samples is an important process.

It is, though, beset with the difficult decision of how best to demonstrate the differ-ences between normal samples and many different disease stages that exist for eachtype of tissue. Up until now, this choice has been restricted to either giving each student a different, but very similar slide, so that they can get the feel of working with a whole sample, or presenting a single image of one sample via projector or onlinetutorial. Both of these options have severe limitations for teaching in groups. With theintroduction of the Olympus dotSlide system, though, this has now changed – eachstudent can review the same sample and control the position and magnification indi-vidually as if it were a real slide. The tutor can also work with all the students in realtime, showing them how best to analyse effectively, pick out rare cases, or test allstudents on the same slide.

High capacity and automatic throughputMany diagnostic, teaching and clinical trials laboratories are faced with a growingnumber of samples to process and it is therefore more efficient to scan slides in batches and review them later. The Olympus dotSlide system can be fitted with aslide loader, which holds up to 50 slides and is integrated with a bar code reader,giving full traceability and hands-off scanning. This ability also makes it quick andeasy to archive slides, producing permanent records.

Storage and preservationAccess and security

When handling a large number of virtual slides, high-quality, secure and rapidaccess data storage is an important consideration. Olympus has therefore workedwith leading electronic storage providers to establish local, regional and internationaldata access and storage network capabilities. With patients’ samples, privacy is para-mount and the system has therefore integrated security settings giving controlledaccess to the slide image and related data.

The old and the newA clear advantage of electronic storage is that slides can be found and loaded quicklyand easily, enabling users to quickly reopen a slide for further investigation, access slides for reference purposes, or prepare presentations for conferences and lectures.Furthermore, there is no degradation to the sample over time – the slide image willalways be as crisp and clear as the day it was recorded. This is especially advantage-ous when archiving old or rare samples, which can then be made available for every-one as if they had been copied.

B

A

To the futureThe advances of the dotSlide system are based on the versatile nature of Olympusmicroscopes, which leaves it ready to be upgraded to match any future requirements.

What is said for the microscope is also valid for future analysis of digitised speci-men. With the ongoing development of the application software, it is expected that computer-assisted diagnosis software will be available which will help to improvedetection and interpretation of tumours and other disease patterns, leaving the patho-logist in full control of the diagnosis result.

B

Virtual digital slides can be easily accessed and viewed simply with anInternet browser, including all relevantinformation

A

Guided by annotations and ‘views’, students can identify regions of interest(ROIs) easily

A

Image below: Virtual digital slides can be accessed instantly and in a controlled way through localnetworks or via the Internet, enabling the users to discuss samples and findings with colleagues orstudents in real time, compare samples and distribute the same sample to multiple locations, e.g.for evaluation.

TakeawayIncreased efficiencyMicroscopy has advanced rapidly over the last few years in terms of the microscopehardware and the techniques used to produce contrast or markers developed to iden-tify molecules, organelles and cell types. Today, we have a much more accurate wayof investigating cellular and bimolecular processes. Many experiments use multiplesections of a sample to show the distribution of a certain molecule. Analysing thesecan take up many hours of the researcher’s time and block the microscope for otherusers. By scanning the sections on the dotSlide system, the researcher can view theirslide away from the microscope and even perform analysis using dedicated imagingsoftware.

Global discussion

Due to the unique way the dotSlide images are stored and transmitted, a researchercan invite the rest of their group to view slides of interest, either individually or as agroup, whether they are in the same building or situated around the world. The virtualfacility offers controllable access for the user, such that with the correct permissions,they can add and edit files. The system is designed to only transmit the amount ofdata required to provide the desired view and is therefore very quick. The Conferencetool window allows a moderator to set up a conference so that all invited clients cansee and discuss what the speaker is showing, plus it also records every aspect ofthe conference, such as the participants’ names, notes and annotations.

FluorescenceFluorescent markers have become one of the main methods of generating clear

contrast in light microscopy. Furthermore, they have allowed very specific identificationof molecules, enabling ultra-precise localisation and transport studies. These capabil-ities are essential for numerous applications throughout many different research areas.For example, fluorescent markers provide oncology researchers with the ability tofollow specific proteins in normal and cancerous cells, to clearly display differencesin translation levels, location or transport. In pharmacological studies, fluorescenceprobes have enabled researchers to study the direct effect of compounds on cellsand whole tissues, accelerating preclinical screening. The dotSlide system can beused with fluorescent samples, giving a completely unique perspective – a slide re-corded in full fluorescence can be thoroughly examined without any risk of bleachingor damage to the cells.

B

A

The dotSlide slide scanner can be up-graded to include fluorescence imageacquisition

B

Online conferencing in real time, location-independent

A

8 dotSlide IN PATHOLOGY AND RESEARCH

Tissue microarraysThe ‘microarray’ concept is rapidly developing, giving both high throughput and

high content for all molecular ‘-omes’ such as the genome, proteome and metabolome.Arrays are a very cost-efficient way of running highly statistically relevant investigationsand, importantly, the array principle applies just as well to tissue samples, known astissue microarrays (TMAs). TMAs consist of many small tissue cores with a defineddiameter that are fixed on a single object carrier. With TMAs, researchers have apowerful way of preparing and staining thousands of different tissue samples underthe same conditions and analysing them at once. This has made them an increasinglyimportant and efficient tool for molecular biological research, pharmaceutical drugdiscovery, gene expression and therapeutic antibody research. The functionality ofthe dotSlide system can be expanded with a dedicated TMA module, facilitatingacquisition and simple analysis of tissue microarrays. This module enables documen-tation of each core separately, accurately recording its slide and core reference fortraceability.

Multifunction

The dotSlide system comprises a research-level microscope with a colour-perfectdigital camera and advanced software, making it a platform with great flexibility, whichallows the addition of a range of accessories, from new objectives to fluorescenceillumination units.

Specialised analysisThe dotSlide software enables simple measurements such as perimeters, distancesand areas without the need for scaling. With additional software tools, a number ofanalysis functions are available to aid in the rapid investigation of the virtual slides.For example, the family of software can perform phase analysis to automaticallycalculate the share of specific tissue types or to identify structures according to aseries of criteria such as size, shape and intensity.

C

Selection or exclusion of TMA cores forscanning with minimum effort

C

Gallery view and multi-image display

Images courtesy of UniversitätsklinikumSchleswig-Holstein, Institut für Pathologie,Prof. Dr. med. A.C. Feller

C

dotSlide IN RESEARCHLife science research has relied heavily on advances in microscopy to move ourknowledge forward. The dotSlide system represents such a step forward, offeringexcellent throughput for extensive image analysis and superb documentation oftissue sections, cell cultures and even tissue microarrays.

Selection of participants and images to bediscussed for online conferences

A

dotSlide – FUNCTIONS AND SYSTEMLeveraging potential Secure image and user management, straightforward creation of Web image galleries, annotationfunctions and the fast and easy navigation make the dotSlide system a versatile platform fordiagnosis, teaching and online conferences. The system consists of robust and carefully selectedhardware integrated with the Olympus BX upright microscope, offering the user peerless reliabilityand flexibility.

10 dotSlide – FUNCTIONS AND SYSTEM

dotSlide – THE FUNCTIONSThe real power of the dotSlide system lies in its functionality. It is designed toprovide all the features needed to make both diagnostic and research use aseasy as possible. These functions are presented to the user via an intuitive andeasy-to-use interface.

Region definitionTo define the area to be scanned, the user can identify a region of interest (ROI)

themselves, using the simple on-screen tools, or leave the system to automaticallydetect the extent of the sample or samples.

Keeping focusedTo make the scan process as fast as possible, the system generates a focus map

using the fast software-based autofocus. Different maps are used depending on thetype of sample being scanned, providing the best solution whatever the sample. Nu-merous coordinates are selected to create the map, which gives the system an instantfocus reference when scanning the slide.

Always sharpFor evaluation of distinct structures, such as the shape of cell nuclei or complex

specimens with different types of tissue, the extended focal imaging (EFI) feature canbe used to produce a perfectly focused image with greatly increased depth of focusat any point within the virtual sample. This is achieved by taking a Z-series of imagesthrough the sample and combining them, in focus elements of each other, into onecrisp image.

High speedIncluding the generation of the focus map, it takes less then 2 minutes for the dotSlideto scan a 10 x 10 mm sample area using a 20x objective with a 0.63x camera adapter.

C

B

AUser-defined regions of interest (ROIs) formore detailed scanning at higher magnifi-cations

A

Different selectable focus maps ensureclear and sharp images, whatever thespecimen

B

C

Smear-stained Papanicolaou, 40x magnificationNon-EFI image (left) and EFI image (right)

Aspect of whole rat embryo, 20x magnificationNon-EFI image (left) and EFI image (right)

C

12 dotSlide – FUNCTIONS AND SYSTEM

Perfect imagesA number of other processes ensure that the images produced are of the utmostquality. Autoscaling, contrast and brightness control, background and shading cor-rection, filters, false colour display and auto-exposure all combine to acquire premiumimages and subsequently modify the display optimally. The system configurationsused for each virtual slide are stored with the slide file for future reference and con-sistency.

Immediate reviewThe image is displayed without delay on the screen whilst being scanned, and

can be immediately edited and viewed without affecting the acquisition. This is dueto the high-performance and unique multi-threading Olympus software. Annotationscan be immediately added to the file in the form of shapes, arrows and text, whilstaudio commentary and an automated scale bar can be displayed in the image. Ad-vanced processing technology allows images to be scanned with high magnificationsas quickly as the equivalent image from a 2x objective.

Easy navigationThe multifunction screen not only displays the digital slide image, but also rele-

vant data and an overview image that can be used for navigation, along with buttonsfor x and y movements and zooming.

File managementWith such high-quality images being produced for each slide, file storage and accessis a very important consideration with the dotSlide system. The first unique step usedby dotSlide is on-the-fly lossless compression, which means that a 15 x 15 mm samplescanned with a 40x objective is compressed from 30 GB to 3 GB for storage. Secondly,with the dotSlide system fully prepared for mass data transfer and storage, it can easilybe integrated with ultra-high capacity storage systems, for which Olympus works withthe leading companies in the electronic storage market.

Access anywhereVirtual slides can be accessed via a number of different methods and can also bepublished to Web databases in a special file format, to enable very easy teleconsult-ation or international collaboration. The system ensures the ideal properties for pro-cessing, data transfer and image display, since parameters such as resolution areautomatically adjusted to what is needed for the user’s monitor. In this way, there isno time delay between changing the view and seeing the image.

B

A

Image serverThe virtual slide image is stored with all the relevant information in a proprietary

format (.vsi), and can be uploaded to a dedicated software environment, which enables instant, controlled, secure access from any local or networked computer, oracross the Internet. The dotSlide system supports different methods of viewing theimage files, enabling viewing permissions to be set and accessed via specialisedsoftware or a standard browser.

The system also enables the virtual slides to be ‘exported to Web’, enabling them tobe placed on a website for teaching or reference purposes. Similarly, the virtual slidecan be recreated on DVD so that it can be distributed to colleagues and studentswithout the need for specialist viewing software.

SecurityPasswords and privileges can be set by the main user so that team members andcollaborators can have differential access to the files. This means that changes canonly be made by authorised people and that they are tracked within the software,which will help investigators maintain traceability.

C

The slide can be reviewed during the scan process

A

The navigation window allows easy orientation and navigation within the slide

B

Clear user and image management

C

14 dotSlide – FUNCTIONS AND SYSTEM

In the frameThe dotSlide system is based on the upright BX Olympus research microscopes, whichenables a range of modules to be easily added to meet the desired specification.

ObjectivesThe objectives used with the dotSlide system are all from the advanced OlympusUIS2 range, offering excellent transmission across the entire spectrum. All objectivesare plan-corrected, displaying high colour fidelity with optimised contrast to visualisestained specimen in front of a bright, natural background. They are available as eitherplan apochromats (UPlanSAPO) or plan fluorites (UPlanFL). The motorised nosepieceintegrates fully with the system for fast, hands-free objective changes.

IlluminationThe 100-watt halogen light source provides the perfect illumination for high-fidelity

brightfield observation and can be polarised using the correct filters. In addition, afluorescent source can be directly fitted to the microscope for axial illumination. Thestability and quality of the light provided by these illumination systems coupled withthe imaging camera produce unmatched colour reproduction, giving users the qualityof image via high-end microscope optics with the convenience of a computer monitor.This is essential for confident case diagnosis and slide interpretation.

High-power controlA top-specification PC-based workstation provides all the power required for com-plex control and processing of both microscope and analysis functions. The large,high-resolution monitor is driven by a dedicated graphic board and a multiformatDVD burner provides easy-to-use optical storage.

Accurate movementA high-precision X, Y stage gives the system excellent accuracy and repeatability,

and can handle standard as well as large slide formats.

Slide handlingThe robustly designed slide loader contains five trays, each holding 10 slides.

The integrated bar code scanner reads all common code formats and can be used to automate slide data entry.

C

B

A

dotSlide – THE SYSTEMThe dotSlide system is built on the highly flexible Olympus ‘optical bench’ micro-scope concept, enabling each system to be exactly right for every requirement.Every component is made to the highest standard and ready for any task.

A dotSlide cameraBest resolution and natural colourreproduction

B Motorised scanning stageFast and precise

C Slide loaderCareful, reliable slide handling

Postfach 10 49 08, 20034 Hamburg, GermanyWendenstraße 14–18, 20097 Hamburg, GermanyPhone: +49 40 23773-0, Fax: +49 40 23773-4647E-mail: microscopy@olympus-europa.com

The manufacturer reserves the right to make technical changes without prior notice. www.olympus-europa.com

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Fully automatic virtual slide scanner

Specifications

dotSlide BX microscope frame

Objectives included: 2x PLAPON, 10x, 20x, 40x UPLSAPO, other objectives may be added optionally, e.g. 100x oil

Illumination: brightfield with 100 W halogen lamp house, optional: fluorescence, polarisation

Slide holder for 1” x 3” slides and for 2” x 3” slides

Multistage insert for batch scanning of four 1’’x 3’’ slides (optional)

Slide loader for 50 slides 3’ x 1’, with integrated bar code scanner (optional)

Digital CCD camera: 2/3” CCD camera, 6.45 µm x 6.45 µm pixel size, high sensitivity, high resolution, Peltier cooled, dynamic range of 3 x 12 bit

dotSlide workstation with Dual XEON™ technology and 500 GB data storage capacity, network interface 100/1,000 Mbit/s Ethernet 4 GB RAM and high-resolution TFT monitor

Data storage solution (optional)

Dimensions and weight (H x W x D), excluding workstation and monitor: system w/o slide loader: 50 cm x 45 cm x 48 cm, 30 kg; system incl. slide loader: 50 cm x 112 cm x 48 cm, 80 kg

CE compliance

Resolution

High-resolution scanning (standard 1x camera adapter): 0.32 µm/pixel (20x/NA 0.75) and 0.16 µm/pixel (40x/NA 0.9)

Fast scanning (0.63x camera adapter option): 0.51µm/pixel (20x/NA 0.75) and 0.26 µm/pixel (40x/NA 0.9)

Scanning speed

10 x 10mm sample area with 20x objective:

• Optional: fast scanning mode < 2 min

• High-resolution scanning < 3 min

Image acquisition

Automatic tissue finding

Seamless stitching

Single button for automatic scanning of up to 50 slides (with optional slide loader)

Focus points are taken automatically on the sample area. Different focus map algorithms are selectable for an optimal result with different types of samples.

Automatic image montage

Software functions and modules

View images locally, zoom, crop and rotate images

Compress images and save images in different file formats

Insert text annotations and drawings; create views of regions of interest, record audio annotations (sound card, microphone and speakers required)

Net Image Server software for remote access to virtual slides with dotSlide software, OlyVIA viewer or Web browser, management of virtual slides and user access to images

Conferencing module for simultaneous and synchronised viewing of virtual slides, conference access control and user management, minute taking tool, chat function

TMA module to acquire individual images of TMA cores

Analysis

Integrated measurement functions: distances, areas, circumferences, point count, export results to Microsoft Excel

Analysis with software packages, e.g. phase analysis to measure shares of tissue types, particle analysis to identify content of tumour cells

Olympus recommends HP for advanced storage solutions

Data compression takes place during the scanning process without any time delay. Algorithms: JPEG, lossless JPEG, JPEG 2000 or no compression.