Multi-Channel Fluorescence Solution for Virtual Microscopy

Multi-Channel Fluorescence Solution for Virtual Microscopy

Enhancing the Capabilities of Virtual Microscopy

The Olympus VS110 Virtual Microscopy System combines microscopy seamlessly with digital imaging, allowing the creation of a virtual slide that is a digital copy of the original specimen. The virtual slide can be shared in real time with multiple persons across the globe. The VS110 generates a high resolution image of the whole specimen, which can be viewed and analyzed from the overview image at low magnification up to maximum magnification by simply panning and zooming. All samples can be evaluated instantly and simultaneously as they are stored electronically on a central server. As a result, the VS110 is ideal for use in a number of areas including research, bio/pharma and education.

The Olympus VS110 can be extended to scan fluorescence images. Such a ‘virtual fluorescence system’ can be used with fluorescent samples, giving a completely unique perspective – a slide recorded in full fluorescence can be thoroughly examined without any risk of bleaching or damage to the cells.

Microscopy has advanced rapidly over the last few years in terms of the microscope hardware and the techniques used to produce contrast or markers developed to identify molecules, organelles and cell types. Today, we have a much more accurate way of investigating cellular and biomolecular processes. Fluorescent markers have become one of the main methods of generating clear contrast in light microscopy. Furthermore, they have allowed very specific identification of molecules, enabling ultra-precise localization and transport studies. These capabilities are essential for numerous applications throughout many different research areas. Many experiments use multiple sections of a sample to show the distribution of a certain molecule. Analyzing these can take up many hours of the researcher’s time and block the microscope for other users. By scanning the sections on the VS110 fluorescence system, the researcher can view their virtual slide image data away from the microscope and even perform analysis using dedicated imaging software.

All VS110 fluorescence system components are designed to interact seamlessly, producing a fully automated, highspeed multi-channel fluorescence scanning system with excellent flexibility and simple operation. These components ensure that users gain the best results for all types of specimens with minimum effort. All users are able to review the same sample, yet control the position and magnification individually, as if it were a real slide. This ability to acquire complete slides at high magnification and resolution virtually, delivers significant benefits to researchers, as well as students in professional education.

VS110 Fluorescence Acquisition Wizard

VS110™ Fluorescence system

A multi-labled sagittal section of a rat embryo. Fluorescence image courtesy of Dr. Dragan Maric, NINDS, National Institutes of Health.

The VS110 fluorescence solution

The real power of the VS110 software lies in its functionality. It is designed to provide all the features needed to make research use as intuitive as possible. These functions are presented to the user via an intuitive and easy-to-use interface. The user is guided step by step through the virtual slide acquisition process by the Scan Wizard. Full-slide scanning can be set up with just three mouse clicks. With the VS110, it is also possible to review the overview slide images and assign individual magnifications, scan areas and other parameters to the slides, even in a batch scan. Although several modes are predefined, the advanced software allows experienced users to be in full control of the details of the scanning process. Therefore, different scan settings can be assigned to individual slides, saving significant scanning time, as well as offering the possibility of only scanning areas of interest, reducing the data file size. For scanning fluorescence images the scan mode is enlarged by a special Fluorescence tab, which has been adapted to fulfill the special requirements of multi-fluorescence microscopy. The acquisition process starts with generating a brightfield or fluorescence overview image of the entire slide. It includes the automatic detection of the specimen to avoid timeconsuming scanning of areas where there is no sample on the slide. As a second step, the high-magnification scan is automatically performed over the whole slide or over smaller, predefined areas of interest using the preselected objective.

Multi-Channel Fluorescence

Configured with high-speed, 8 position excitation and emission filter wheels, the VS110FL provides increased flexibility in the choice of fluorochromes as well as extremely fast acquisition of multi-channel images. Individual configuration of each of the color channels helps to ensure the optimum focus for each channel. Image acquisition is performed using a high performance cooled 14bit monochrome CCD camera and stored as full 16bit grayscale data.

Virtual-ZTM for Fluorescence Imaging

The VS110 fluorescence system is also capable of scanning multiple large specimens in up to 15 z-planes. By selecting the scan mode, the reviewer can just simply focus through the sample, as well as examine regions of interest in different dimensions. At each position of the whole sample, the VS110 system acquires a series of fluorescence images at various focus positions, and these fluorescence z-stacks are stored within the images and can be focused virtually on the monitor. The image is displayed without delay on the screen while being scanned and can be immediately viewed without affecting the acquisition. This is due to the highperformance and unique multi-threading Olympus software.

Advanced Features

The optional VS-Desktop Review Station software contains advanced imaging tools to further enhance the acquired virtual slide images. Included in these tools are smoothing and sharpening filters, min mean and maximum projections, as well as no neighbor and nearest neighbor deconvolution. Additional tools in VS-Desktop include separation of each of the color channels, as well as exporting this data to other 3rd party software for further image analysis.

General VS110™ Specifications

Fully automated virtual slide scanner

• Motorized BX® microscope frame
• Objectives included: 2x PLAPON, 10x, 20x, 40x UPLSAPO, other objectives may be added optionally
• Illumination: brightfield with 100 W halogen lamphouse
• VS110-S1 with slide holder for one 1” x 3” slide
• VS110-S5 with slide holder for five 1” x 3” slides or for two 2” x 3” slides
• VS110-L100 with slide loader for 100 3” x 1’’ slides, with integrated bar code scanner
• Digital CCD camera: 2/3” CCD camera, 6.45 μm x 6.45 μm pixel size, high sensitivity, high resolution
• Peltier-cooled, 14-bit dynamic range
• 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)
• CE compliant

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 camera using 0.5x camera adapter: 0.34 μm/pixel (20x/NA 0.75) and 0.17 μm/pixel (40x/NA 0.9)

Scanning speed (brightfield)

• 15 x 15 mm sample area with 20x objective: standard scanning mode < 3 min

VS 110 Fluorescence Features

• Dual cameras support brightfield and fluorescence
• 14-bit monochrome camera (XM10)
• Support of 16bit grayscale data
• Multi-channel imaging (> 3 channels)
• Dedicated fluorescence acquisition wizard
• incl. monochrome or color overview acquisition and 10x overview image option for greater detail
• incl. auto exposure for optimal exposure time
• Virtual-Z™ fluorescence acquisition
• Optional: advance filtering eg deblur, max intensity projection (available in optional VS-Desktop Software)
• Optional: oil scanning up to 100x

Image acquisition

• Acquisition scanning wizard
• Automatic sample and tissue finding
• Autofocus and manual focus routines
• Multilayer scanning: multiple scans in one image
• Seamless stitching
• Virtual-Z™ acquisition
• Single button for automatic scanning of up to 100 slides (with VS110-L100)
• Reviewed batch scan
• Scan directly to .vsi, .tif or .btf formats

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 VS110 software, OlyVIA viewer or Web browser and management of virtual slides and user access to images
• Conferencing module for simultaneous and synchronized viewing of virtual slides, conference access control and user management, minute-taking tool, chat function
• TMA module to acquire individual images of TMA cores, create virtual TMA’s
• NetImage Server and OlyVIA™ can support Hamamatsu .ndpi file format

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 tumor cells

Dimensions (mm)

* This measurement may vary according to interpupillary distance. The measurement given pertains to a distance of 62 mm.