Volume 34 Issue 4, Fall 2007, pp. 431-436


Virtual microscopy (VM) has been implemented and evaluated in the histology and general and systemic pathology courses at the University of Iowa Carver College of Medicine. Advantages of VM over traditional microscopy include accessibility and efficiency of learning and the ability to integrate VM with computer-assisted interactive learning. Advantages of using VM as opposed to digital photomicrographs include the ability to pan and zoom, explore the slide, and make independent observations. Although VM is used in a case-based format for teaching histopathology to medical students at the University of Iowa, VM may also be effectively implemented in other medical-student teaching models, including integrated and problem-based learning curricula and the classical pathology laboratory. Additional Iowa venues and courses using VM teaching include pathology of human disease for bioscience graduate students, cytology education, a comparative pathology research resource, and histology and histopathology for veterinary medicine. This article reviews the history and evolution of VM in medical pathology and its implementation at Iowa.

As the twentieth century closed there was increasing pressure, for reasons of both financing and use of space, to remove microscopes from the medical curriculum and replace them with digital photomicrographs and computers. Pathology and histology educators feared that one outcome of this paradigm switch for students might be a loss of the ability to explore microscope slides by panning and changing magnifications and to make independent observations. Fortunately, in the 1980s and 1990s, the ability to create giant, high-resolution facsimiles of whole microscope sections was being developed.

Although the technology to create virtual slides as giant montages of multiple microscopic fields of view was described more than a decade ago,1 ,2 the necessary computer processing speed and RAM to acquire and manipulate single giant image files in desktop computers was not readily available until approximately 1996. Another significant milestone was the development in 1996 of an open-source tiled file format for giant multi-resolution pyramidal flashpix (FPX) by Kodak and others, which provided the technical ability to stream giant image files over the Web in a standard Web browser. At about the same time, an early prototype flashpix file server was developed by Live Picture/MGI. a

In 1997, the authors considered the above information and sought a way to emulate the pedagogical advantages of the traditional microscopy using desktop computers and the Web. After developing a prototype using MicroBrightField (MBF) virtual slide acquisition and MGI Zoom flashpix converter and server software,b we obtained a grant from the National Library of Medicine (NLM) with the goals of (1) implementing and evaluating virtual slides in our histology and pathology courses, (2) modeling virtual microscopy for other institutions and teaching environments, and (3) developing a database of virtual slides for other medical schools to use.

Beginning in 2000, we have progressively implemented and evaluated the use of virtual slides in our first-year histology and second-year pathology courses.3–6 Using the University of Iowa data set as a foundation, we have added slides from other institutions, thus creating a not-for-profit Virtual Slidebox of histology and pathology that can be used by other educators.

This implementation also created an infrastructure for the generation and evaluation of other courses and databases at Iowa, including (1) a Virtual Laboratory for the annual American Association for Cancer Research Pathobiology of Cancer Workshop;7 (2) a Cervical Cytology Slidebox education and testing project (under development);8 (3) a National Center for Research Resources (NCRR) grant–supported Virtual Slidebox of Comparative Cancer Pathology (under development);9 and a Virtual Slidebox of Veterinary Histology and Histopathology (under development). All the above programs can be accessed from the University of Iowa Virtual Slidebox home page at <http://www.path.uiowa.edu/virtualslidebox>.

Acquisition and viewing technology for virtual slides is presented in detail elsewhere;3–7 the acquisition and delivery technology that we have used in our laboratory will therefore be only briefly reviewed. Also note that, subsequent to the early developments described above, a very large variety of virtual-slide file formats and servers has been developed, both proprietary and non-proprietary, by a now rapidly expanding virtual-slide industry.

Virtual slides have been acquired in our laboratory by either a Virtual Slice system purchased from MBFc or, more recently, a ScanScope system purchased from Aperio.d After editing to enhance image brightness, contrast, and sharpness, the original files have been converted to a variety of multi-resolution pyramidal file formats, including FPX,e PFF,f and SVS.g The predominant format for slides currently used on our sites is the SVS JPEG compression file format, because it is an open-source format and can be converted to any other format. We also have plans to convert our data set to the Composite WebSlide (CWS) format,h which is compatible with the Bacus WebSlide server.i Since the initial files have been captured and saved as open-source files, we can convert them to any file format compatible with a viewer that suits the educational need. We are currently serving and viewing our virtual slides with software purchased from MBF; an annotation applet has also been purchased from MBF. Our virtual-slide sites are developed and optimized for the PC running Microsoft Internet Explorer; they also will work on a Macintosh.

All of our virtual slides are maintained in a Perl-scripted MySQL database. This database, developed in-house at Iowa, is integrated with the server, viewer, and annotator. Each virtual slide contains data-entry fields for species, organ, diagnosis, slide contributor, and free text (descriptions, pathogenesis, and correlations). In addition, there are links to JPEG images (gross and radiological) and other relevant Web sites. Also contained in the database are coordinates of arrows, text, and the ability to bring up other virtual slides in a split frame. By means of on/off buttons in the database editor, the virtual slides, together with associated data, are made to appear in one or more of the educational programs. Also scripted from the database is an additional program, the Comparative Search Tool, which allows any of the more than 900 virtual slides and their associated data to be viewed in side-by-side frames, where they can be independently manipulated. The advantage of this tool is the ability to compare, side by side, normal versus disease slides, disease processes from organ to organ, and disease processes across species.

The following is an overview of current implementation at the University of Iowa. More details can be found in previous publications.3–9

Virtual Histology Laboratory

Prior to 2000, the histology laboratory at the University of Iowa was taught in traditional format. A printed laboratory syllabus contained descriptions of the glass slides and the entities the student was expected to identify on glass slides in a traditional microscope laboratory; there were 110 glass slides in the student slide boxes. Faculty presented a pre-lab with photomicrographs. In the microscope laboratory, if students had questions about a slide at their single-headed microscopes, they raised their hands and an instructor would assist each of them individually. Progress exams in the course were administered with photomicrographs, and the final examination was administered using glass slides.

In a formative evaluation carried out in two units of the course in 2000, students rated the virtual laboratory significantly better than the traditional laboratory with respect to both accessibility and efficient use of their time.3, 5, 6 Students felt that the virtual laboratory slides were at least equal in image quality to the traditional glass slides; they also commented that “the move around and zoom in and out function is like looking at the real microscope.” However, some students wanted “arrows to structures that were difficult for an inexperienced viewer to locate” and “a mechanism to quiz our progress.” As a result of this feedback, all course slides were subsequently digitized, and links were made to gross and Visible Human imagesj and to atlases of annotated photomicrographs. These resources could be accessed from within or outside the institution. When the virtual histology laboratory was fully implemented in 2001, course content and the laboratory schedule remained the same, with the exception of using virtual slides rather than photomicrographs in the pre-lab. During regularly scheduled laboratories, students now have the option to attend a computer laboratory, a traditional glass-slide-and-microscope laboratory, or a combination microscope and computer laboratory. After the first week of class, students gravitate almost exclusively to the combination laboratory. The instructor who continues to staff the laboratory frequently interacts with a group of students at a computer terminal on which faculty and a number of students can simultaneously observe the field of view on the virtual slide or at one of several multi-headed microscopes in the laboratory. In spite of heavy use of virtual microscopy for teaching, evaluation of student performance has remained consistently high on practical examinations, both with photomicrographs in unit examinations and with traditional glass slides and a microscope in a comprehensive final practical examination.

In 2003, in response to student requests, we added (1) arrows with descriptions that point out the features a student is expected to identify and (2) quizzes for each unit, which consist of exercises that ask students to identify the tissue and then a follow-up question requiring them to identify key elements of the tissue, indicated by arrows. This is the most popular portion of the histology Web site. Based on the number of hits on the site, these quizzes seem to have markedly increased the use of the virtual histology laboratory, especially before examinations. It should be emphasized that students are still tested using glass slides and microscopes at the end of the course.

Virtual Slides in Pathology Courses

The University of Iowa teaches pathology differently from many other medical schools: there is a two-semester stand-alone pathology course with a weekly two-hour small-group session called Case Analysis, preceded by flexible study time in the microscope laboratory. To prepare for Case Analysis, students are given four weekly unknown cases, consisting of a case history, physical findings,; laboratory data, radiological images, a microscopic slide, and gross images. Approximately 70 cases are discussed over the duration of the course. Students study the cases independently, and in the weekly small-group session they are expected to be ready to discuss any aspect of any of the cases. Each small group consists of seven to eight students and one pathology faculty or resident facilitator. Primary presentation of the pathology is done by the students rather than by the facilitator.

Prior to the implementation of virtual slides in 2000, students were given the case summaries in a syllabus. During preparation for small-group sessions they had to examine the glass slide with a traditional microscope and view gross and radiological images on mounted photograph displays in the pathology microscope laboratory. In small-group sessions, students displayed the glass slides by video-microscopy, and the gross and radiological images were displayed as 2 × 2 slides using a carousel projector.

In the fall of 2000, all the glass slides for the Case Analysis cases were digitized and linked to all other course materials, which were also posted on the Web.k The use of microscopes and glass slides in preparation for case discussion subsequently fell precipitously, from nearly 100% in 1999 to ∼25% in 2000, ∼10% in 2001, and < 5% in 2002, with a reciprocal increase in use of virtual microscopy4,.6—despite the fact that glass slides and microscopes were still available to students. In small-group meetings, some faculty were initially adamant that students demonstrate with a traditional microscope and video projection; now, however, all faculty have very willingly accepted virtual slides, and all students demonstrate pathology in their small groups using virtual slides and a computer projector. Subjective evaluations by facilitators and students indicate that virtual slides are a very efficient and accessible way to learn pathology. In end-of-course subjective evaluations, faculty report no decline in students’ morphologic skills in small groups. Also, according to comments made at the yearly course-evaluation meeting, many faculty feel that students come to Case Analysis small groups better prepared to discuss and present histopathological findings than when they studied from glass slides with traditional microscopy. Subjective evaluations completed by students also point out the following advantages:

  1. Virtual slides are always in focus, with proper lighting and condenser adjustment.

  2. The image quality is much better than with video-microscopy and nearly a good as looking through the ocular of a traditional microscope.

  3. Virtual slides allow the presenter to show a whole-mount overview of the slide, which is not possible with traditional microscopy or video-microscopy.

Pathology of Human Disease for Graduate-Level Bioscience Students

Based on successful implementation of virtual slides in medical student histology and pathology courses, we implemented virtual slides in the yearly American Association for Cancer Research (AACR) Pathobiology of Cancer Workshop.7 In this course, non-physician pre-doctoral students and post-doctoral fellows who work in cancer research spend an intensive week learning the morphological, clinical, and molecular aspects of human cancer. During the workshop, students examine approximately 100 glass slides in microscope laboratories. Our intent in implementing virtual slides was not to replace traditional microscopy in the workshop; the course faculty believe that traditional microscopy is integral to the training of bioscience students. In 2001, we implemented virtual slides in one of the course units in order to evaluate virtual slides as a teaching modality in facilitating student learning at the traditional microscope. We found that demonstration by faculty with virtual slides instead of with photomicrographs before students examined the glass slide enhanced students’ ability to grasp morphologic features on the glass slides. Following this initial formative evaluation, virtual slides were implemented in all units of the course, and they continue to be used with success. Because the organization of the AACR workshop laboratory is very similar to that of the traditional pathology laboratory at many US medical schools, this implementation may serve as a model for medical and veterinary pathology courses with a similar teaching format.

In recent years, progressive advances in scientific knowledge have dictated an escalation in academic offerings of cutting-edge molecular-based courses for the non-professional graduate-level student. Students have aptly expanded their enrollment in these new programs, but often at the expense of more conventional courses such as basic anatomy and morphology.10 Furthermore, there is a declining faculty base trained to teach many of these same courses.11 We believe that VM technology can be a bridge to engage graduate students’ interest while providing a labor-saving forum for faculty instruction. To this end, in addition to continuing the AACR workshop laboratory, we are developing a graduate-level curriculum in comparative histology and animal models of disease, using VM technology to further enable faculty (both at the University of Iowa and elsewhere) in training the biomedical scientist.

Cytology Education

VM technology, which has been widely implemented for histology and histopathology education, is by nature two-dimensional (2D), capturing the glass slide content only in the x and y axes, with a single level of z-axis focus. Although 2D viewing works well for histological sections, it is problematic for cytology because of the three-dimensional (3D) nature of cells and clusters of cells in cytology specimens. Thus, implementation of VM in cytology education has been slow to develop.12–14

Funded by a grant from the National Library of Medicine, the authors have been studying the effectiveness of VM for education and testing in cervical cytology, using both 2D and novel 3D (x, y, and z axes) virtual-slide technology developed by MBF. The project was also designed to allow (1) evaluation of the effectiveness of virtual microscopy in cytology teaching and education; (2) comparison of the value of 3D technology with traditional 2D VM technology; (3) evaluation of the diagnostic accuracy of virtual slides versus real glass slide in cervical cytology interpretation; and (4) the development of a public-access Web site for teaching cytology students. Seventy-nine paid volunteer cytologists and cytotechnology students participated in the evaluations.8 There was a positive consensus among the evaluators that virtual cervical cytology slides would be a useful augmentation to education and testing. We found that diagnostic accuracy using virtual cytology slides was similar to that for glass slides (94% vs. 96%) and that there was no difference in diagnostic accuracy between 2D and 3D slides (p = 0.28). Nevertheless, the ability to focus 3D slides in the z axis was strongly endorsed by the participants because of the uncertainty and frustration of having some cells out of focus on 2D virtual slides. Finally, 3D over the Web is too slow if one is to examine the entire slide, but 3D cytology is very compatible with delivery via DVD, especially when downloaded to the desktop. An example of an application of 2D cytology over the Web can be seen at <http://www.path.uiowa.edu/virtualslidebox/cervical_cytology>.

Clinical laboratory glass smears and wet mounts from microbiology and urinalysis are further examples where 3D VM may have a role to play. The application of 2D technology in hematology is less problematic;15 however, some hematology bone-marrow smears also require 3D technology for adequate viewing.

A Comparative Pathology Research Resource

The Department of Pathology at the University of Iowa has been funded by the National Center for Research Resources of the NIH to develop a Virtual Slidebox of Comparative Cancer Pathology. The goal of this Web-accessible research-related resource is to expand the current knowledge base of the biomedical research community concerning individual cancer sub-types across species. It will also serve as a resource for investigators to compare specific spontaneous, induced, and genetically engineered animal cancers with human cancer and with normal tissues.9 A pilot unit on lymphomas can be viewed at <http://www.path.uiowa.edu/virtualslidebox/cancer_pathology/index.html>.

This Web site displays cancers from humans and domestic animals (e.g., dog, cat) in addition to laboratory animals, thus demonstrating for the research community the breadth and diversity of lymphomas in vertebrates. It is hoped that the site will increase an awareness that may lead to new routes of investigation not yet explored. In addition to increasing our knowledge about human disease, it will also contribute to cancer research in a clinical veterinary medicine setting.

Histology and Histopathology in Veterinary Medical Education

An implementation of VM in a veterinary curriculum using the WebSlide file format from BacusLabs is described in another article in this issue of JVME.16 We at Iowa, in collaboration with the Department of Veterinary Pathology at Iowa State University in Ames, IA, have also initiated an open-source Web site for teaching veterinary histology and histopathology, which includes contributions from several institutions.l A screen shot from the database is shown in Figure 1. This layout is very similar to that of the human Virtual Slidebox of Histology and Virtual Slidebox of Histopathology.

Figure 1: Screen shot of a slide from the Virtual Slidebox of Veterinary Histology and Histopathology illustrating the menu for endocrine disorders at the left, with a feline islet amyloid slide selected. The panel on the left indicates the contributor (Peter Brown, DVM, PhD, of the University of Bristol, UK), and radio buttons bring up annotations and arrows for selected fields of view on the slide. A navigation window in the upper right-hand corner allows the student to pan and zoom the entire slide. A whole mount of the actual glass slide scanned is at the upper left.

Although this resource is in a very early stage of development (with only 96 virtual slides to date), we believe that, once completed, it will have the potential for the same national and international impact as our NLM-funded human histology and histopathology Web sites.

Public Access to the Virtual Slide Database

To accomplish the third goal of the NLM grant, publicly accessible Virtual Slidebox of Histology and Virtual Slidebox of Histopathology Web sites have been developed, incorporating material from multiple medical schools, so that the database now includes more than 264 virtual histology slides and more than 637 virtual histopathology slides.m The content on the histopathology site is based in part on a list of core morphologic entities for medical students that we created in 1998.17 The virtual slides on these two Web sites have links to JPEG images of gross normal and pathology specimens, radiological images, Visible Human images, and descriptive text. This site allows faculty to (1) use the Virtual SlideBox of Histology or Histopathology on a computer as they would use any box of slides for student labs or faculty demonstrations; (2) incorporate the source code of individual virtual slides into HTML pages of their own Web-based programs; (3) take screen shots of any field of view from the slides in the Virtual Slidebox and use them in their own Web pages and PowerPoint presentations; and (4) acquire the data set of virtual slides and deliver them from their own server/viewer software. The virtual slides are not for sale, but course directors, may acquire the NLM grant–supported virtual slide files via removable media. Some fair-use and licensing restrictions apply to use of the database content, and human use restrictions forbid commercial use. The Virtual Slideboxes of Histology and Histopathology have experienced widespread national and international use, as evident from thousands of hits on the Web sites from around the world as well as requests from over 25 medical schools for our virtual-slide data set. These medical institutions have used the data set to develop histology and histopathology educational software applications of their own, which they may then augment with virtual slides of their own study sets, either by purchasing their own slide scanner or by having the slides scanned commercially.

Course evaluations at the University of Iowa indicate that both students and faculty favor the use of virtual slides over a traditional microscope, glass slides, and video-microscopy. This result is due mostly to the markedly increased efficiency and accessibility of virtual microscopy over traditional microscopy, with no apparent decrement in learning. Virtual microscopy has also been implemented in pathology and histology courses at many other institutions with success and findings similar to those at Iowa.18–23

If the current trend continues, the implementation of VM may eventually result in medical students’ not using the microscope at all in histology and pathology courses. This potential outcome raises the question of whether physicians in training need to use a microscope. We believe the answer is an unqualified “yes.” Physicians need to know how to use a microscope to examine urine sediment, gram stains, and possibly blood smears. However, they do not need to use the microscope to examine the hundreds of slides needed to cover the content of histology and pathology courses; this can be done much more efficiently via the computer, with virtual slides. One might also ask whether, if and when microscopes are not available for training in histopathology, digital photomicrographs might not be as good as more expensive virtual slides. Though no comparison study has been done to answer this question, we believe the answer is “no,” because virtual microscopy allows students to explore a section, find structures independently, discover relationships and variations, and draw conclusions. Thus, students become active rather than passive learners, just as they do when they use real slides and the microscope—but more efficiently.

Finally, the use of virtual slides, though initially an expensive proposition, may eventually become less expensive than traditional microscopy, especially if the space for a traditional microscope laboratory is converted to other uses. In our institution, we have calculated the cost of a microscope laboratory for 50 students to be about $100,000 per year, which approaches the complete start-up costs for VM, including the purchase of a virtual slide scanner.

Our faculty initially expressed concern that virtual slides and the computer would decrease faculty–student interaction. These fears have since evaporated. Pathology and histology faculty in our courses continue to play significant roles in student learning, but they now do so in a more interactive and stimulating manner. The role of pathology faculty in facilitating our unique Case Analysis sessions has not changed, while in histology, working together with students in a small-group setting, faculty are able to use a more Socratic approach that leads students to explore structure/function relationships and clinical correlations. Our histology faculty and graduate assistants no longer spend excessive time fielding repetitive questions from individual students. This new approach results in a more satisfying and more efficient method for learning for all.

In addition to the venues described above, virtual slides can be used to replace or augment glass slide sets, video-microscopy, and digital photomicrographs used in a wide variety of other teaching formats, including (1) interdisciplinary problem-based learning, (2) continuing medical education, (3) proficiency testing, (4) certifying exams, (5) computer-assisted instruction, and (6) adaptive learning and testing strategies that cannot be implemented with real glass slides.

We believe that the results of implementation of virtual microscopy at the turn of the twentieth century, at the University of Iowa and elsewhere, suggest that in the twenty-first century virtual slide technology can be successfully implemented in teaching laboratories, including veterinary histology and pathology courses, where traditional microscopy and photomicrographs are currently used.


a See <http://www.mgisoft.com>.

b MicroBrightField Inc., Colchester, VT 05446 USA <http://www.mbfbioscience.com/>.

c MicroBrightField Inc., Colchester, VT 05446 USA <http://www.mbfbioscience.com/>.

d Aperio Technologies, Vista, CA 92081-8545 USA <http://www.aperio.com/>.

e Eastman Kodak Co., Rochester, NY 14650-0948 USA <http://www.kodak.com/>.

f Zoomify, Inc., Santa Cruz, CA 95061 USA <http://www.zoomify.com/>.

g Aperio Technologies, Vista, CA 92081-8545 USA <http://www.aperio.com/>.

h Aperio Technologies, Vista, CA 92081-8545 USA <http://www.aperio.com/>.

i Bacus Laboratories, Inc., Lombard, IL 60148 USA <http://www.bacuslabs.com/>.

j Visible Human Server, École Polytechnique Fédérale de Lausanne, Switzerland <http://visiblehuman.epfl.ch/>.

k See <http://www.path.uiowa.edu/cgi-bin-pub/vs/case_analysis/cases.cgi>.

l See <http://www.path.uiowa.edu/virtualslidebox/vet_histopathology/index.html>.

m See <http://www.path.uiowa.edu/virtualslidebox>.

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