ARTICLE

Creating a Histology–Embryology Free DigitalImage Database Using High-End Microscopy andComputer Techniques for On-Line BiomedicalEducationVICTOR W. SILVA-LOPES AND LUIZ H. MONTEIRO-LEAL*

The development of new technology and the possibility of fast information delivery by either Internet or Intranetconnections are changing education. Microanatomy education depends basically on the correct interpretation ofmicroscopy images by students. Modern microscopes coupled to computers enable the presentation of these imagesin a digital form by creating image databases. However, the access to this new technology is restricted entirely tothose living in cities and towns with an Information Technology (IT) infrastructure. This study describes the creationof a free Internet histology database composed by high-quality images and also presents an inexpensive way tosupply it to a greater number of students through Internet/Intranet connections. By using state-of-the-art scientificinstruments, we developed a Web page (http://www2.uerj.br/�micron/atlas/atlasenglish/index.htm) that, inassociation with a multimedia microscopy laboratory, intends to help in the reduction of the IT educational gapbetween developed and underdeveloped regions. Anat Rec (Part B: New Anat) 273B:126–131, 2003.© 2003 Wiley-Liss, Inc.

KEY WORDS: database; video microscopy; digital imaging; microscopy; medical education; virtual microscope; computer-assisted learning; CAL

INTRODUCTION

The understanding of histology andembryology is a process that startswith the correct interpretation of mi-croscopy images, which leads to a bet-

ter comprehension of morphologyand the morphogenesis of cells, tis-sues, and organs. However, until re-cently, the access to this visual infor-mation by students was limited toperiodic and short practical micros-copy sessions backed up by textbooksand printed atlases. The limited num-ber of microscopes in the medical andbiological institutions of underdevel-oped areas was an added problem.Moreover, in most universities, beforethe practical classes of histology andembryology, the histological prepara-tions were presented by the professorwith a micro-slide projector. Becauseof poor resolution and illumination,the projected images were deliveredwith poor quality, hampering the cor-rect visualization of the samples bythe students.

In the 1980s, the advent of video-microscopy revolutionized histologyclasses by remarkably improving theimage quality (Inoue and Spring,1997). Nevertheless, despite theabove-described advances, some diffi-culties still remain: the costs of video-

microscopy systems are usually pro-hibitive for many schools inunderdeveloped countries and the re-stricted number of light microscopesconsequently limits the access of thestudents to a greater quantity of visualinformation.

The availability of the Internet andof advancements in computers cou-pled with microscopes created untoldpossibilities for improving histology,embryology, and anatomy courses(Trelease et al., 2000; Heidger et al.,2002). These advances will probablyhelp to close the educational gap be-tween developed areas and those thatare in the process of catching up, bymaking robust information technol-ogy (IT) infrastructure and opportuni-ties more available. In addition, thereis the possibility of direct access tolight and electron microscope imagesthrough the creation of image data-bases and the transmission of sucharchives to several sites by Intranetand Internet. This access allows stu-dents from different institutions orthose with a limited number of micro-

Mr. Silva-Lopes is a Ph.D. student at theMicroscopy and Digital Image-Process-ing Laboratory from the State Universityof Rio de Janeiro. He works with thecreation, implementation, and impact ofdigital techniques in the teaching of his-tology and embryology. Dr. Monteiro-Leal is Professor of Histology and Em-bryology at the State University of Riode Janeiro, is the Head of the Micros-copy and Digital Image-Processing Lab-oratory, and works with cell motility andalso with the creation of computer pro-grams to analyze cell morphology andmorphometry.*Correspondence to: Dr. Luiz H. Mon-teiro-Leal, Universidade do Estado doRio de Janeiro, Depto. de Histologia eEmbriologia, Laboratorio de Microsco-pia e Processamento de Imagens, Av.Prof. Manoel de Abreu, 444 3° andar,Maracana Rio de Janeiro, RJ, 20550-170Brazil. Fax: �55-21-22689874; E-mail:mleal@uerj.br

DOI 10.1002/ar.b.10021Published online in Wiley InterScience(www.interscience.wiley.com).

THE ANATOMICAL RECORD (PART B: NEW ANAT.) 273B:126–131, 2003

© 2003 Wiley-Liss, Inc.

scopes or unable to acquire a sophis-ticated video-microscope system toeasily download images developed bya state-of-the-art equipped center.Also, the unlimited and plastic virtualspace, contrasted with the greatly lim-ited, expensive, and fixed space avail-able for pictures in books, introducesa vast amount of information to thestudents (Brinkley et al., 1997).

However, a new step for microanat-omy could take place by bringing to-gether the new techniques and proto-cols of image processing for the

production of single image montages.By using the high-resolution framesobtained by wide-aperture light mi-croscope lenses and the high-qualitymagnified electron microscope im-ages, it is possible to create montagesshowing larger microscopic fields ofview with greater resolution (Mon-teiro-Leal et al., 2003; Romer et al.,2003).

This study sets out how to developand assemble a high-quality histologydatabase to be used by the studentsand by creating a multimedia labora-

tory, with the association of com-puter, Intranet or Internet, micro-scopes, and television, a less-expensive way to present theinformation. The impact of this workon the Brazilian and internationalcommunities are also reported.

METHODOLOGY

Preparation of the HistologicalSections

Healthy Wistar rats were killed in aCO2 camera (following the directionsset out by the Ethics Committee foruse and care of experimental animalsfrom the Biological Institute of theState University of Rio de Janeiro).

Their organs and tissues were ex-tracted, quickly finely sectioned,washed in saline buffer, and then pre-pared by following the specific proto-cols for light and electron microscopy,as described before (Karnovsky, 1965;Stoward and Pearse, 1980; Bancroftand Stevens, 1996; Monteiro-Leal etal., 1996; Bozzola and Russell, 1999).

Digital Microscopes

The three microscopes (light, scan-ning, and transmission electron mi-croscope) were connected to and con-trolled by computers. The imagesvisualized in the light microscopes,using high numerical aperture and ab-erration-free objectives, were after-

Figure 1. Schematic representation of the major steps concerning the construction of thehistology database. After the acquisition of high-resolution images in the digital micro-scopes (LM, light microscope; TEM, transmission electron microscope; SEM, scanning elec-tron microscope), the frames are transferred to the central computer (central computer)and a digital image montage (digital montage) is created. The computer is used to createthe database and is connected to other computers (host computers) by Internet orIntranet.

By using the high-resolution frames

obtained by wide-aperture light

microscope lenses andthe high-quality

magnified electronmicroscope images, it is

possible to createmontages showing

larger microscopic fieldsof view with greater

resolution.

ARTICLE THE ANATOMICAL RECORD (PART B: NEW ANAT.) 127

ward acquired with the help of a dig-ital camera (SoundVision SV-Micro).The pictures obtained by using thescanning electron microscope weredigitized in the machine itself, and theones observed in the transmissionelectron microscope were acquiredwith the associated slow-scan cooledCCD camera (Proscan elektronischeSysteme, Scheuring, Germany) on axiscamera (Hiller et al., 2000; Campanatiet al., 2002; Monteiro-Leal et al., 2003).

Central Computer

To process, analyze, and perform theimage montages, a central computerwas assembled and the KS 400 (Zeiss-Vision, Jena) and the SIS-Auto (SIS-SoftImage, Munster) image process-ing systems were installed. Thiscomputer was a Pentium III, with 512MB RAM memory, and 20 GB diskspace connected to a network workingat 2 MB per second.

Software

The Web site was created by using thebasic HTML-creator software that ispart of Netscape software (NetscapeComposer). The images were furtherprocessed and compressed with PaintShop Pro 6.0 (JASC Software, Inc.,USA. 1999), and some montages werecreated with the use of the MIA –module from SIS-Auto and the Ipde-luxe image processing software(Wootton et al., 1995; Monteiro-Lealet al., 2003).

ACQUISITION, MONTAGE, ANDPRESENTATION OF DIGITALIMAGES

To obtain high-quality digital images,our microscopes were connected to acentral computer that stored all im-ages transferred by Intranet (Figure1). The central computer (CentralComputer, in Figure 1) hosted the da-tabase and the mother Web site, a mir-ror of the published Web site, locatedin the main computer of the Univer-sity. The images produced by thethree microscopes were improved andcompressed using the specific soft-ware (described in the Methodologysection) in the central computer. Dig-ital image montages were prepared(Digital Montage in Figure 1) to pre-vent poor resolution associated withimages acquired with enlarged field ofview, using, for example, low-magni-fication light microscope objectives;and the acquisition process by digitalcamera, in the case of the transmis-sion electron microscope (Inoue andSpring, 1997; Campanati et al., 2002;Monteiro-Leal et al., 2003). By the useof high numerical aperture objectivesin light microscopy or by the use ofhigh magnification with the transmis-sion electron microscope, high-qual-ity images were prepared that, on theother hand, negatively present asmaller field of view. The montage ofseveral sequential acquired images(digital montage in Figure 1) by thecomputer solved the above-mentionedproblem about the limited field ofview of these images, resulting inhigh-quality frames with enlargedfield of view (Figure 2A,B; Monteiro-Leal et al., 2003). Afterward, the im-ages were then transferred by Internetor Intranet to the host computers(host computers in Figure 1).

Figure 2. Digital montages created by assembling several high-resolution microscopeimages. A: The result of the montage of 200 single high-resolution, small-field images in afinal high-resolution, large-field frame. This picture shows a 14-day-old mouse embryo. B: Thesame procedure, this time performed with electron transmission microscopy frames. Thepicture shows a high-resolution, large-field montage of a lung section obtained after thecomputer association of 16 single frames. Scale bars � 1 mm in A, 3.5 �m in B. [Color figurecan be viewed in the online issue, which is available at www.interscience.wiley.com.]

128 THE ANATOMICAL RECORD (PART B: NEW ANAT.) ARTICLE

IMAGE COMPRESSION ANDPREPARATION OF THE WEB SITE

When image montages were created,their higher quality had the drawbackof producing huge archives; some filesgreater than 80 MB (TIFF files). Thesearchives were too heavy to be trans-ferred between computers by Internet.To solve this problem the images werecompressed into JPEG files using thesoftware listed in the Methodology sec-tion. The initial high resolution of theimages, as described above, compen-sates for the partial loss of resolutioncaused by the compression process.

Afterward, the images were up-loaded in the Web site using NetscapeComposer and linked serially to aprincipal page that shows the optionsmenu. The access to the Web site(http://www2.uerj.br/�micron/atlas/atlasenglish/index.htm), called DigitalAtlas of Histology (Figure 3A–D), wasfree. First-time users were requestedto fill out an electronic questionnaire,which was afterward used to collect

information regarding the users. TheAtlas was prepared in Portuguese andEnglish, and accompanying each im-age was explanatory text. It waslaunched on-line in 1999 and since2001, a new-access counter showedthat there have been more than 35,000visitors, an average of 3,000 to 4,000guests per month.

DIGITAL ATLAS USER PROFILE

The profile of the visitors to the DigitalAtlas, obtained by the on-line ques-tionnaire (10,500 questionnaires), isanalyzed in Figure 4. Part A displaysthe percentage of regional access inBrazil, which as expected, representsa very high degree of access (90%). Itshows that users in the Southeast re-gion performed more than 50% of theaccesses and, together with the South-ern region, represented 70% of all ac-cesses in the country. These regionsare the most developed in Brazil. PartB shows the same analyses under-taken in A, but refers to “international

access.” From Europe came 39% ofthe accesses, followed by North Amer-ica with 25% (USA corresponded to70% of the accesses) and South-Amer-ica with 22% (the Brazilian accesseswere not computed). Guests from Asiacorrespond to only 7% of the accesses.Part C presents an analysis of theabove-mentioned data concerning us-ers’ educational status or background.The data shows that 52% of the guestsare university students, followed bygraduates and high-school students.

MULTIMEDIA LABORATORY

The digital histology database was ei-ther accessed at home individually orthrough computers in libraries or inany other public place (data notshown). Moreover, the image data-bank was used during the normalpractical classes in the microscopelaboratory. To make these new tech-nologies available to all students andto improve the content of the lectures,the following methodology was intro-

Figure 3. Sample images obtained from the Digital Histology Atlas. A: The menu page from where the other 350 English pages can beopened from the Atlas. B: An example of the short explanatory texts related to the images. C: A light microscope montage that opens onetissue image database. D: One sample from the several scanning electron microscope images that can be found in the Atlas. [Color figurecan be viewed in the online issue, which is available at www.interscience.wiley.com.]

ARTICLE THE ANATOMICAL RECORD (PART B: NEW ANAT.) 129

duced in the practical sessions. Be-sides the video-microscopy used bythe professors at the beginning of theclasses to show what preparationswould be taught on a specific day, theday’s theme could be also reiteratedby the use of the high-quality imagesobtained by light and electron micro-scope and stored in the Digital Atlas.To do this, a computer was connectedby Intranet with the central computer(where the databank is situated), andby using a switch, the images of the

Digital Atlas were presented on thesame television monitor, which is alsoconnected to the video-microscope(the same one that had been previ-ously used to show the day’s prepara-tions). The computer was equippedwith a fast Internet connection andwith a video board with analog out-put. A less costly design was devel-oped for a small educational unit innorthern Brazil, where a computer withInternet was connected to a 34-inch TV.It was possible, with the help of such a

system, to use the Digital Atlas throughthe Internet instead of the more expen-sive video-microscopy equipment, alsoexcluding the necessity for histologicalslides. Alternatively, some modern tele-visions are equipped with a specific in-put for directly plugging into a com-puter, functioning as a computermonitor. In such cases, one can use anyregular computer, avoiding the use of avideo board with analog output.

FINAL CONSIDERATIONSMaking Internet access and computertechnologies available to an increas-ing number of students is a great chal-lenge for the future. The opportunityto use these advances in education, inparticular in biomedical education,should be a priority. Some publishedpapers are concerned about the im-pact of the new technologies on edu-cation (Brinkley et al., 1997; Clark,2001; Lin and Hsieh, 2001; Hallgren etal., 2002; Pear and Crone-Todd, 2002;Vogel and Wood, 2002; Yazona et al.,2002), but one question still remains,Will these tendencies diminish thegap between the developed and under-developed areas or will they increase it?

The purchase and use of high-endequipment in scientific research is notuncommon, even in some regions ofunderdeveloped countries. However,having access to the high-quality im-ages produced by such sophisticatedequipment is not an easy task. Usu-ally, students have infrequent contactwith a light microscope and very lim-ited experience with electron micro-scope images, often confined to thosefound in textbooks. This situation iseven worse in less-developed regions.Owing to resource limitations, bothfinancial and material, some univer-sity courses cannot afford to purchaseeither microscopes or video-micro-scopes to be used in the practical his-tological and pathological classes.

To present information in a moredemocratic way, allowing the biomed-ical student or even the average citi-zen to have access to high-quality “sci-entific-type” image, a free histologicaldatabase was developed. To accom-plish that, digital controlled micro-scopes were used in association withdigital image processing software andIntranet or Internet. In a first step, theproduction of digital image montages(as shown in Figure 1), allowed the

Figure 4. Graphic distribution of the Digital Histology Atlas. A: The percentage of access in2001, separated by regions in Brazil, shows that the great majority of guests (70%) came fromthe South and Southeast, which are the most-developed regions of the country. Studentsfrom Brazil represent 90% of all accesses. B: The same analysis as in A but refers to thedistribution of international visitors. Europe has the greatest access majority, followed byNorth America (the students from the United States represent 70% of all accesses) and SouthAmerica (the guests who accessed from Brazil are not included). C: Profiles (educationalbackground) of the guests who accessed the Atlas during the past year. The data showthat the great majority of visitors were college students.

130 THE ANATOMICAL RECORD (PART B: NEW ANAT.) ARTICLE

visualization of high-resolution wide-field frames (Figure 2). The advantageof such montages is that they help inthe observation of detailed structures(normally observed only at highermagnification) and associated details,cells or tissues that are normally visiblein low-magnification images (Woottonet al., 1995; Monteiro-Leal et al., 2003).The students easily associate the mon-tage viewed on the computer monitor,or on the television, to the morphologydescribed by the professor in the theo-retical lectures or previously read inbooks, but now, as mentioned before(Vichitvejpaisal et al., 2001), with a farsuperior quality, when compared withthe normal textbook images.

The production of a free Web-basedDigital Histology Atlas (Figure 3), or-ganized with images created by theuse of state-of-the-art equipment andcomputer montages, is allowing stu-

dents from different regions to ac-quire greater amounts of visual infor-mation. The students have accessedthe Atlas using personal computers inthe university, in their homes or inpublic places, such as Internet cafes.

On the other hand, as confirmed bythe graphics in Figure 4, our worksuggests that the Web-based distanceeducation is not bringing about a re-duction in the learning discrepanciesbetween the developed and the under-developed regions. This finding is re-flected by the incomparably greaternumber of visitors to our Web sitewho come more from developed re-gions, both in Brazil (regions Southand Southeast) and the rest of theworld (the access from Europe plusUSA and Canada totaled 64% of theforeigner visitors to the Atlas). Thelimited number of computers and In-ternet servers in less-developed re-

gions could explain this finding. Con-cerning biomedical education, thissituation, instead of diminishing it,could amplify the gap, because theprofessional’s ability would be influ-enced by the quality of the universi-ties’ equipment and Internet facilitiesfrom which he/she graduated.

One possible solution to the aboveproblem is the association of a com-puter with Internet connections to thesame television monitor used for thepresentation of the images observedby video-microscopy. This set-up wasused in the practical biomedicalclasses and was shown to be less ex-pensive than buying several comput-ers and connecting them to the Inter-net. Moreover, we have found that,when using a computer with video-output and a 29- to 36-inch television,there is no need for expensive dataprojectors and image quality is notgreatly diminished. The implementa-tion of such a system in medicalschools located outside the main citieswould result in a broader normaliza-tion of biomedical education regardingthe histology, anatomy, embryology,and pathology classes, thus diminishingthe learning gap between these placesand the more developed centers.

ACKNOWLEDGMENTSThe authors thank Jonas Dias deBritto Filho for technical support,Helmut Troster for critical review,and Ana Francisca Miranda for En-glish revisions. The present work wassupported by Capes and CNPq.

LITERATURE CITED

Bancroft JD, Stevens A. 1996. Theory andpractice of histological techniques. Edin-burgh: Churchill Livingstone.

Bozzola JJ, Russell LD. 1999. Electron mi-croscopy: Principles and techniques forbiologists. Boston: Jones and BartlettPublishers.

Brinkley JF, Bradley SW, Sundsten JW,Rosse C. 1997. The Digital Anatomist In-formation System and its use in the gen-eration and delivery of web-based anat-omy atlases. Comput Biomed Res 30:472–503.

Campanati L, Holloschi A, Troester H,Spring H, Souza W, Monteiro-Leal LH.2002. Video-microscopy observations offast dynamic processes in the protozoonGiardia lamblia. Cell Motil Cytoskeleton51:213–224.

Clark GT. 2001. Education problems andweb-based teaching: How it impacts den-tal educators? J Am Coll Dent 68:25–34.

Hallgren RC, Parkhurst PE, Monson CL,Crewe NM. 2002. An interactive, web-based tool for learning anatomic land-marks. Acad Med 77:263–265.

Heidger PM, Dee F, Consoer D, Leaven T,Duncan J, Kreiter C. 2002. Integratedapproach to teaching and testing in his-tology with real and virtual imaging.Anat Rec 269:107–112.

Hiller SA, Probst W, Seybold V, Zellmann E,Kabius B, Trondle A. 2000. New frame-transfer wide-angle slow scan CCD cam-era allows recording of distortion-freeimages for digital montages. Microsc Mi-croanal 6(Suppl 2):1020–1021.

Inoue S, Spring K. 1997. Why video? In:Video microscopy: The fundamentals.2nd ed. New York: Plenum Press.

Karnovsky MJ. 1965. Formaldehyde-glut-araldehyde fixative of high osmolarityfor use in electron microscopy. J CellBiol 27:137A.

Lin B, Hsieh C. 2001 Web-based teachingand learner control: A research review.Comput Educ 37:377–386.

Monteiro-Leal LH, Farina M, Souza W.1996. The free movement of Tritrichomo-nas Foetus in a liquid medium: A video-microscopy study. Cell Motil Cytoskele-ton 34:206–214.

Monteiro-Leal LH, Troster H, CampanatiL, Spring H, Trendelenburg M. 2003.Gold finder: A computer method for fastautomatic double gold labeling detec-tion, counting and color overlay in elec-tron microscopic images. J Struc Biol141:228–239.

Pear JJ, Crone-Todd DE. 2002. A social con-structivist approach to computer-medi-ated instruction. Comput Educ 38:221–231.

Romer DJ, Yearsley KH, Ayers LW. 2003.Using a modified standard microscopeto generate virtual slides. Anat Rec (NewAnat) 272B:91–97.

Stoward PJ, Pearse AG. 1980. Histochem-istry, theoretical and applied. Edinburh:Churchill Livingstone.

Trelease RB, Nieder GL, Dorup J, HansenMS. 2000. Going virtual with QuickTimeVR: New methods and standardisedtools for interactive dynamic visualisa-tion of anatomical structures. Anat Rec(New Anat) 261:64–77.

Vichitvejpaisal P, Sitthikongsak S,Preechakoom B, Kraiprasit K, Parakka-modom S, Manon C, Petcharatana S.2001. Does computer-assisted instruc-tion really help to improve the learningprocess? Med Educ 35:983–989.

Vogel M, Wood DF. 2002. Love it or hateit? Medical students’ attitudes to com-puter-assisted learning. Med Educ 36:214–215.

Wootton R, Springall DR, Polak JM. 1995.Image analysis in histology: Conven-tional and confocal microscopy. Lon-don: Cambridge University Press.

Yazona JMO, Mayer-Smith JA, RedfieldRJ. 2002. Does the medium change themessage? The impact of a web-based ge-netics course on university students’ per-spectives on learning and teaching.Comput Educ 38:267–285.

The production of a freeWeb-based DigitalHistology Atlas is

allowing students fromdifferent regions to

acquire greater amountsof visual information.

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