Using Virtual Slides with the MBF Virtual Slice System

Introduction and Background

Use of a traditional microscope and glass slide has long been the teaching method of choice for medical education in many subjects, including pathology, histology, physiology, and embryology. Traditionally, students use a microscope to examine normal and diseased tissues and cells, which are preserved on glass slides and viewed at various levels of magnification. However, there are several problems inherent in this approach, including limited access to microscopes, preparation of multiple similar specimens for students, the cost of the microscopes themselves, and issues of classroom access. Efforts have been made to digitally capture microscopic images for instructional purposes, but the limitation of this method is that only one field of view at one magnification is captured, the overall sense of the scale of the tissue is lost, and there is no ability to navigate through an entire tissue sample.

MicroBrightField, Inc. has developed a creative solution to the problems of traditional and digital microscopy education with the development of the Virtual Slice Module to their popular Neurolucida™ program, which captures morphological information contained on glass slides, digitizes the information, and delivers it over the Web. This bioinformatics system allows users to collect images of complete microscopic specimens at the highest magnification of a light microscope and stitch these images together into a single large image, called a “virtual slide”. These very large montages are stored in web-enabled databases from which they can be shared with colleagues and students via streaming images over computer networks. The system provides solutions not only to the challenges of teaching with microscopes, but also to issues faced by scientific and clinical collaborators.

The technologies incorporated in these virtual slides are described here, including the image acquisition system, the software used to seamlessly integrate contiguous sections, the database storage system, the web-enabled viewer, and the interactive annotation system that can be used to place labels on areas of interest on these virtual slides.

Benefits and Practical Applications of the Virtual Slide

Research Applications

Slides accessible from anywhere at any magnification: The Virtual Slice System allows researchers to create Internet accessible virtual slides and databases of virtual slides that can be viewed at any magnification at any time and from any remote site with network or Internet access. The virtual slides surpass the limitations of field size of traditional optical microscopy by providing low power views that encompass an entire specimen, while allowing the detailed examination of the highest optical magnifications through dynamic zoom capabilities. Collaborating researchers can share an entire slide or set of slides over the Internet, a vast improvement over static photographs or images of single fields of view.

Interactive interface: The framework and interface of Virtual Slice is designed so that users can indicate certain locations on the virtual slide at specific magnifications that are linked to comments or questions. This interactive capability can be customized for real-time, open access, discussion by groups of collaborating researchers. Investigators can access the same histological sample, and “point” to areas of interest while carrying on a conversation on-line, or archive comments for other users to view at their convenience. Labels of text and arrows may be overlaid on the virtual slice to facilitate the collaborative process.

Access to more diverse specimens: In research, one of the great limitations is access to a large sample population. Through the use of virtual slides, which can be analyzed with any of the MicroBrightField analysis products (Neurolucida™ or Stereo Investigator™), researchers can access a large number of specimens from diverse sources (e.g.: various species, strains, ages, preparations, sexes) for comparison. Sharing of material via virtual slides can also be used to increase sample size while limiting the number of animals needed to be sacrificed.

Private networks available: Researchers have the option of setting up their own private intranets to distribute images to colleagues privately. A local network can be used to distribute slides within an institution, or a limited access secure slide database can be set up for restricted viewing over the Internet.

Archiving of rare and precious biological material: Vast numbers of biological specimens are prepared on slides, but much of this material degrades over time – samples fade, glue dries, and glass can crack. There is tremendous benefit in putting the original material on-line in permanent digital databases. Other researchers would have access to rare or time-consuming preparations without unnecessary duplication of effort. With the virtual slide database, users from around the world can share the very best specimens, making the highest-quality and rarest preparations available. In addition, the use of single specimens by a number of researchers will ultimately decrease the amount of animal tissue necessary. Histological specimens taken from animals that are now protected are in existence, and could be used to learn more about these endangered species if they are made public as part of a virtual slide database.

High quality preparations available: Novice technicians and investigators trying new histological preparations often do not know what to expect in terms of the quality of the final product. In our experience as scientific consultants, we have seen many sub-standard preparations being used because the user simply did not know that better results were possible. With a freely available database of virtual slides, users will be able to see the expected outcomes of a variety of preparation methods, including the common flaws (e.g. uneven shrinkage, torn tissue, uneven staining) of certain methods, in addition to top quality preparations.

Public archives: As technology improves, the architecture of the Virtual Slice bioinformatics system will make it possible for granting institutions to request that all grant awardees that produce slides for their research voluntarily make them available to a public archive. Researchers in different locations can access the same microscopic images and share their experimental data and observations. Password-restricted access can be set up for users wishing to share sensitive material only with approved viewers.

Education Applications

Slides accessible from off campus locations at any magnification: Our system allows instructors to create Internet accessible virtual slides and databases of biological virtual slides that can be viewed at any magnification at any time and from any remote site with Internet access. The magnifications available exceed the limitations of field size of optical microscopy. Access to lower power views encompassing the entire specimen lets students see the “bigger picture” of the entire section, as well as the location of the region of interest within the section. Detailed examination of the highest optical magnifications is enabled with dynamic zoom capabilities, which can give a larger picture of the most intricate details.

Slides accessible at any time: A major consideration for microscopy labs is the limited access that students have to the microscopes and slide collections. Virtual slides are accessible any time a computer and Internet connection are available, so that students can study from their own homes or dorm rooms on their own schedules.

Interactive interface: We have designed a framework and interface so that instructors can easily design their own on-line tutorials. Instructors can indicate certain locations on the virtual slide at specific magnifications that are linked to instructive text or quiz questions. Users can customize this interactive capability for restricted access by instructors only, or real-time open access for remote discussion groups by students, instructors and/or teaching assistants.

Students have access to more diverse specimens: Students of bioscience at all academic levels are now able to examine specimens of virtual slides without the need for a microscope. This vastly increases the amount of histological material available to students, and thereby enhances their learning. One of the most difficult lessons to impart to students of biological science is the vast degree of normal variation between healthy specimens. This lesson can be demonstrated by accessing virtual slides of the same tissues from a number of different sources.

Private networks available: Teachers and researchers also have the option of setting up their own private intranets to distribute images to colleagues and students. A local network can be used to distribute slides for courses among students and instructors.

Archiving of rare and precious biological material: Vast numbers of biological specimens are prepared on slides, but much of this material degrades over time. There is tremendous benefit in putting the original material on-line in permanent digital databases. Rare or time-consuming preparations will be available without unnecessary duplication of effort, while students would have access to specimens of uncommon cases from around the world. Tissue specimens from normal and pathological human tissue can be shared between universities and medical centers, minimizing the need for handling of potentially hazardous human materials. With the virtual slide database, users from around the world can share the very best specimens, making the most high quality and rare preparations available for everyone to use for study.

Save on the cost of microscopes: The cost of classroom microscope and glass slides make it prohibitive for many lower level science courses to allow students the experience of viewing slide material, and at all levels this cost is a significant consideration. The availability of high resolution virtual slide databases will make it possible for biological, anatomical, and histological education courses at all levels to dispense with classroom microscopes. This can significantly diminish the cost of a school’s laboratory operation, and open the teaching of microscopic materials to institutions formerly unable to afford the microscopes and slides necessary for these subjects.

The Technology

Image Acquisition: We have developed an innovative method to create images of microscopic specimens that are composed of thousands of fields of view. The basic hardware required for the creation of virtual slides is a computer microscopy system composed of a research microscope, a high-resolution stepper motor stage and focus controller, a color CCD video camera, a frame grabber, and a PC with monitor.

The requirements of the acquisition PC deserve special mention here, as large digital images require computers with fast CPUs and a large storage capacity. The CPU used should be the fastest possible, we recommend at least a Pentium IV CPU running at 1.3 GHz or faster with a minimum of 1 GB of RAM. A large and fast hard drive (40 GB minimum) is also recommended. An archival/backup device such as a tape driver or CD writer is also recommended. Small numbers of images can also be acquired at the MicroBrightField facilities for users not wishing to invest in acquisition hardware.

Our acquisition software allows the user to define graphically the region of interest of the microscope slide at low magnification, and to acquire the image at high magnification. If the region of interest is larger than a single field of view, the software automatically drives the motorized stage to access the entire defined region. Once the user defines the area of interest, the software controls the motorized stage to scan the slide at high magnification with sub-micron positioning resolution, and assures that every location within the defined area is imaged.
The software has several important features for accurate and seamless data acquisition, including: 1) A merging function, which automatically “stitches” the images together into a composite image, which is saved as a single file; 2) Several auto-alignment functions that will automatically align images collected from adjacent fields of view; 3) An automatic background correction function to eliminate the effects of non-uniformities in the microscope’s illumination; and 4) An optional auto-focus module based on contrast levels obtained from a Z axis scan. In cases of thick sections, the user can manually select the correct focus for each image field.

Database Storage: We have developed a user-interface for a web-enabled, relational database. This database contains information about the virtual slides, such as: species, sex, age, staining, and disease state. The database for these images can reside on a central server, in essence operating as a “virtual slide box”.

Once a virtual slide has been acquired, it is converted to a FlashPix format file. In addition to being ideal for web viewing, this format is increasingly supported by a number of commercial imaging programs. FlashPix uses a hierarchical multi-resolution structure known as an image pyramid. The pyramid structure allows for rapid access to image data at any level of resolution and any spatial selection. The structure of the FlashPix file lets FlashPix-enabled applications select the appropriate resolution for a given operation and process only the portion of the image needed. In the case of a virtual slide, a web browser can display a low-resolution version of an image for fast downloading, then zoom into the area to be shown at higher resolution.

The virtual slides as acquired are extremely large images, easily exceeding 1.5 GB (more than 1500 fields of view). The FlashPix technology is used in conjunction with JPEG compression to reduce the file size of the full images for transfer through the Internet. While viewing, the user-defined region of interest will be less compressed so that no visible loss of image quality will occur. This smart decompression and smart transfer technology is of major importance to the virtual slide system. Images the size of a virtual slide would take hours to download if the entire file were sent at one time. However, by streaming only the information requested by the user, the amount of data transferred remains relatively small, allowing for fast downloads, as well as real-time ability to scan through large areas of tissue at multiple magnifications with high resolution.

Our database is structured to meet the demands of handling these large images as well as contain rapid querying and security capabilities. Due to the large amount of image information stored in the database, the architecture is extensible so that it can reside on several image servers cooperating with one central database. In order to make images rapidly accessible, we use a Pentium Xeon multiprocessor server with 1 GB RAM running. The database server runs the Windows 2000 Server operating system. The database server is placed on the Internet, and uses the Tomcat web server from the Apache group to serve dynamic content.

Users wishing to host their own database should have a server with speed of at least 866 MHz, with at least 1 GB of RAM and a 60 GB or greater hard drive. The Windows 2000 operating system is recommended.

Visualization: Montages of acquired microscope images have been generated before, but there has not previously been a way to easily transmit and view these images over a network, due to their extremely large file size and network bandwidth limitations. Our virtual slide viewer developed for use with the virtual slide database is interactive and permits the rapid display of any portion of the acquired images at any magnification. The viewer enables users to zoom into designated areas and navigate through the virtual slide with the functionality familiar to a microscopist. The interface is platform-independent, thus allowing PC, Mac, Linux, Unix, etc. users to access the database without the need to download additional viewing components.

Successful visualization of a virtual slide depends on high-performance viewing technology. Users can interactively zoom in and out and view any area of the image at any magnification. Once the user chooses to zoom in, the image server sends only the new region of interest at a higher resolution. This is different from simply using an optical zooming technology in which pixels are viewed as larger; the actual high-resolution image of the selected area is sent to replace the lower resolution overview. The more the user zooms in, the higher the resolution of the image that is sent, to the limit defined by the microscope lens used to acquire the data. The virtual slide allows for a small amount of additional dynamic zoom capacity by using pixel enlargement, allowing for a greater magnification than achieved by the light microscope.

The requirements for viewing virtual slides are far less than those for acquiring and storing them, due to the interactive streaming nature of the viewer, which downloads only the user-selected regions of the slide at highest resolution. For viewing virtual slides, we recommend a 500 MHz processor, with 128 MB of RAM, operating on Windows 95 or higher. The requirements for web browsers differ depending on operating system. On a PC, Microsoft Explorer 4.5 or higher and Netscape Navigator 4.02 are recommended. On a Mac, Netscape Navigator 6.0 or higher is recommended.

Viewer features enhance the usability of our system beyond that of a traditional microscope. We provide a “navigation window” which always displays the low magnification view of the entire image along with information about the location of the current view. Altering the size of the browser window can alter the size of the navigation window to meet user needs. For example, if a user has a slower modem, the use of a smaller navigation window will require the streaming of smaller amounts of information to complete the image area selected.

Annotation capabilities: A significant feature of our virtual slide viewer is its annotation capabilities. The annotation tool allows users to make graphic annotations, such as arrows and text, and to overlay them on any position on the virtual slide. An author can specify properties for each annotation, including color, font, and size. Additionally, the author can designate a specific magnification or a range of magnifications in which the annotations will be displayed. Each annotation contains four components of information: the position, the magnification, the annotations, (such as arrows and text labels) that have been added to the slide, and any notes that the author has entered. Subsequent users who view the slide will see the author’s notes alongside the virtual slide. Clicking on a link from the notes will navigate the slide viewer to the position and magnification that was stored with the annotations, and show the author’s labels.