ANSWER - The term virtual microscopy is used in different contexts. In one context the term virtual microscopy refers to interactive microscope simulators which are graphical representations of a microscope whose settings can be altered to simulate what would happen to an image when the settings of a real microscope were changed. This type of virtual microscope can be a very useful as a learning tool. Good examples of this type of virtual microscope can be found on the Florida State University website.
A different type of Virtual microscopy is a rapidly developing tool which is set to make a big impact in the laboratory. In this context a virtual microscopy is a means of creating and then viewing a virtual slide (sometimes called a digital slide) over a computer network or on the screen of a stand alone computer using special virtual slide viewer software.
It is the virtual or digital slide which is the key enabling component of a virtual microscopy system. The virtual slide is a very large digitised image file of a glass slide which can be viewed, panned and zoomed (while still maintaining real resolution rather than increasing empty magnification) on a computer screen.
A virtual slide in the context of Pathology is the digitised image of a tissue section captured at a resolution which is suitable for diagnostic purposes. Virtual slides can be easily stored, archived, retrieved, annotated, duplicated, distributed, integrated into electronic patient records and viewed over the Internet or private computer network.
It is from these features of a virtual slide that all the benefits of virtual microscopy flow and make many things possible which are not possible with glass slides. With virtual slides it is possible to simultaneously view an image of the whole tissue section and zoom into a particular part of the section for a more detailed view. This can be an invaluable aid to the Pathologist who wants to relate a detailed view of one part of the section to the overall geography of it.
Q2 - How are the virtual slides created?
ANSWER - Virtual slides are created by digital slide scanning systems using mainly area scan or line scan CCD technology to digitise the image of the glass slide. Other technologies are starting to appear but are still in the minority.
- Area Scanning Systems. Area scanning systems often comprise a microscope, a conventional digital camera, motorised XY stage and a Z stepper to adjust focus. A PC with integral stage and microscope control boards automate the stage and stepper and control the microscopeâs illumination, condenser, top lens and objectives. These systems work by moving the slide mounted on a motorised scanning stage under the microscope objective, capturing a digital image of the field of view (often referred to as a tile) and then moving to the adjacent field of view to repeat the digitisation process until the whole or selected region of the slide has been scanned. The tiles produced by this method are then âstitchedâ together using sophisticated software which aligns each tile to create a large mosaic image.
- Line Scanning Systems. These systems often comprise an automatic slide loader, robotic stage, a single very high quality microscope objective and a linear array camera. The slide is scanned in a continuous manner up and down the region of interest to create the virtual slide.
Q3 - What are the pros and cons of the different methods?
ANSWER - As area scan systems are usually microscope based systems the technology used is familiar to laboratory staff and existing equipment could possible be used or adapted for use to create virtual slides.
However, using an area scan system and camera with a resolution of 1280 x 1024 pixels (1.3 Mega pixels), approximately 800 tiles would have to be captured for a tissue section area of 15 mm x 15 mm if a virtual slide resolution of 0.46 microns per pixel is to be achieved. If the desired resolution is 0.23 microns per pixel then four times as many tiles have to be digitised to give this resolution. Needless to say system calibration is critical and the electromechanical and software components have to be of the highest quality to ensure the accurate alignment of possibly thousands of image tiles.
Line scan systems have their origins in machine vision systems therefore the technology is unknown to most Pathology laboratory staff. There is also very little likelihood that existing laboratory equipment could be used or adapted to create virtual slides. Unlike microscope based systems not all of the optical techniques are not yet available on line scan type systems.
Line scan systems produce far fewer tiles than an area scan system. Using the example for the area scan system given above, a linear array sensor with 4096 pixels would produce approximately only15 tiles for an area of 15 mm x 15 mm at a resolution of 0.23 microns per pixel. This greatly reduced number of tiles simplifies the process of tile alignment.
In addition to the reduced number of tiles, linear array systems continuously focus many times along the length of the tiles and may be better able to compensate for subtle changes in the topology of a specimen than area scan systems.
Q4 - What kind of resolution is on offer?
ANSWER- Typically resolutions of 0.23 and 0.46 microns per pixel are possible which equates to resolutions obtained using good quality microscope objectives of 20x and 40x magnification respectively.
Q5 - Can you say more about Time Delay and Integration (TDI)?
ANSWER - This is a specialised sensor readout mode which integrates the light signal from each pixel in the image and can greatly improve the quality and contrast of images even under low light level conditions. For example a 4096 x 64 line scan array would integrate (build up) the signal from each pixel by a factor of 64. This feature can be particularly beneficial when scanning fluorescence labelled slides. The primary advantage of TDI operation is the increased image integration time it gives in comparison to standard linear array sensors.
Q6 - From an optics and photonics standpoint, what are the key technical challenges and how are they overcome?
ANSWER- To be able to consistently create a high fidelity digital representation of the material on the original glass slide. This will be particularly important for virtual slides which are to be used as the source for quantitative data analysis software programs. Experienced microscope users will expect virtual microscope operation to be as intuitive and fast as traditional microscopes. For example, tissues which require the microscope user to focus back and forth through a sample require large amounts of data to be handled very rapidly.
Some of the most serious short term challenges are not directly related to the creation of virtual slides by advanced systems based on line scan technology but more so on the shortcomings of current PC technology. A huge amount of data is generated by some slide scanning systems but the bottleneck for rapidly scanning and viewing slides is often the architecture of the PC.
Q7 - What the main sectors driving this technology?
ANSWER- Routine clinical, research and teaching applications in Pathology and high throughput screening and analysis of slides in the Pharmaceutical industry.
Q8 - What are the big benefits to the user?
ANSWER - The virtual slide is the key enabling component of a virtual microscopy system and allows digital images of whole slides to be easily stored, archived, retrieved, annotated, duplicated, distributed, integrated into electronic patient records and viewed over the Internet or private computer network. It is from these features of a virtual slide that all the benefits of virtual microscopy flow and make many things possible which are not possible with glass slides.
Q9 - What's next for virtual microscopy - for example, future applications and forthcoming improvements in the technology?
ANSWER- Current virtual microscopy systems are used to scan fixed, stained tissues. Perhaps future systems could be used for live, unstained cells using sensors which are tuned for different wavelengths. The high resolution provided by line scan systems might be very interesting in the context of
of time lapse experimentation with growing live cells. Currently researchers use video cameras which produce good and useful images but the intracellular detail obtained is not of a particularly high resolution.
Current applications for virtual microscopy have been aimed at the life science market. However, there is no good reason this technology should not be also used to scan material science slides. The optics and illumination would be different but the applications would be similar.
In the life sciences the major application areas are as follows;
- Routine & Computer Aided Diagnoses in the Pathology laboratory. At present, the interpretation and quantification of disease state by examining a tissue section is dependent on the knowledge and skill of an individual Pathologist. This is a subjective process which is time consuming and with the introduction of an increasing number of diagnostic molecular markers will become even more time consuming and possibly prone to error. The worldwide shortage of Pathologists exacerbates this problem. The ability to produce a virtual slide opens the way for more objective and faster analysis slides by linking to image analysis software to support the decision making process of the Pathologist.
The ability of advanced digital slide scanning systems to automatically scan batches of slides and identify slides by their barcodes will free Pathologists from the more mundane aspects of reporting and should also help to reduce errors. Through a fully digital medium it will be possible to have an integrated approach to patient data collection, distribution, reporting and the seamless integration of different data types – images, text, database, statistics, quantitative analysis – into flexible information management systems.
- Research. The use of Tissue Microarray Analysis (TMA) is now becoming standard practice in the research pathology laboratory. Digital slide scanning systems which can produce a virtual slide of a TMA slide allied with the use of TMA analysis software are significantly increasing the throughput of these slides and enabling data to be extracted and analysed more rapidly and efficiently.
- Education & Training.Virtual microscopy allows viewing of virtual slides by large numbers of students or trainees over a computer network thus avoiding the necessity for them to be at a particular venue at a set time to attend a teaching or training session. This can lead to a large reduction in the time and expense required to organise and run these sessions. Traditionally, the students and trainees would have viewed images generated by a digital camera mounted on a microscope. Each person would have only been able to view the part of the slide and objective magnification selected by the microscope operator. Using web server software it is now possible for each person to view a section of the slide selected by them at the magnification they choose. Not only are there significant cost savings to be made but the quality of the learning experience is enhanced.
With the advent of wireless networks it should be possible to extend the accessibility of teaching and training materials to make them available on portable and pocket computers/devices to facilitate “anytime, anywhere” learning.
- Archiving. The physical bulk of large slide collections have created massive storage and accessibility problems. The creation of virtual slides which can be stored locally on computer systems will allow them to be easily retrieved for retrospective studies.
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