Cytometry 13:109-116 (1992)
0 1992 Wiley-Liss, Inc.
HOME: Highly Optimized Microscope Environment’ Gerard Brugal, Richard Dye, Bruno Krief, Jean-Marc Chassery, Hans Tanke, and J a m e s H. Tucker Equipe de Reconnaissance des Formes et Microscopie Quantitative, CERMO, Grenoble, France (G.B., B.K., J.-M.C.); Medical Research Council Human Genetics Unit, Edinburgh, Scotland, United Kingdom (R.D., J.H.T.); and Sylvius Laboratories, University of Leiden, Leiden, Netherlands (H.T.) Received for publication April 15, 1991; accepted July 31, 1991
The Highly Optimized Microscope Environment (HOME) is a computerized microscope designed to assist pathologists and cytotechnicians in clinical routine tasks. The prototype system consists of a IBM-PC compatible computer and a light microscope in which a built-in highresolution computer display image is superimposed on the optical image of the specimen. Also, a manually operated encoding stage and objective turret encoder are used to provide continuous monitoring of the stage coordinates and microscope magnification to the computer. This allows any position on a slide to be uniquely defined and makes it possible to measure interactively lengths and areas larger than the size of the microscope field. Software, written in the C language and operating under the MS-DOSMS-Windows environment, is
In modern hospitals, the pathology laboratory plays a n essential role in the diagnosis and prognosis of most serious diseases, and therefore in the subsequent medical decision-making and health care for the patient. In spite of this essential role, pathology practice has to date remained relatively untouched by recent developments in information technology: routine pathology is still carried out largely by microscopic examination of specimens by the pathologist, followed by the reporting of his opinion concerning the appearance of the specimen back to the requesting clinician. While this is satisfactory for the majority of specimens, for most types of pathological sample there is a “grey area” containing some specimens for which the result is not clear and is therefore subjective, unreliable, and poorly reproducible [lo]. One approach to improving the diagnosis for such specimens is the introduction of quantitative analysis. Counting is the first quantitative approach which has been used extensively in haematology (differential
controlled by means of a mouse-driven cursor moving over menu light-buttons displayed on the microscope image. The HOME microscope workstation is potentially useful in a wide range of applications such as i) tagging information on particular cells and tissue structures that can thus be accurately located and relocated, ii) performing morphometric measurement, differential counting, and stereological assessment of biological specimens, and iii) training and educating laboratory personnel. Finally, HOME will offer in the near future a userfriendly interface for automatic image processing of cells and tissue entities in interactively selected specimen areas. Key terms: Microscopy, image cytometry, image histometry, computerized microscopy
white blood cell count), tumour pathology (mitotic index), and hormonal cytology (pycnotic index). Several other quantitative techniques for pathology have been investigated extensively for many years. Interactive computer morphometry, in which the user can delineate lengths and areas on a projected microscope image using a digital tablet or similar instrument, has been found to be diagnostically useful in many types of tumour pathology, bone pathology, and muscle pathology 111. Stereology, the use of statistical sampling to estimate geometrical properties of specimens, has also been applied to similar problems [7]. Moreover, densitometry has been very widely used to measure total
‘The project was partly funded by the Commission of the European Communities under its Advanced Informatics in Medicine (AIM) action, Contract A 1007.
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cellular DNA content and other cell constituents [9], and more recently for the color analysis of a variety of immunocytochemical markers [5]. All these measurements can be carried out using image cytometry (either interactive or automatic). However, in spite of these achievements there has been a disappointing impact on pathology practice. These techniques so far have been confined mainly to research laboratories, and are still not used widely in routine pathology. A major reason for this is lack of suitable equipment. Although a number of general-purpose computer morphometry and image analysis systems are commercially available, these are often large and expensive, and require substantial expertise to reconfigure for the pathologist requirements. Also, they are not satisfactory for measurement on complex biological specimens: often the user must oscillate between microscope and computer, first using the microscope and its control to find a suitable field to measure and then having to tu rn to the computer keyboard and screen to carry out the required measurement operations. Usually, this cycle must be repeated many times to obtain a statistically representative value for the overall specimen. For this reason the acquisition of diagnostically relevant information with these systems is often labour-intensive, time-consuming, and poorly correlated with the conventional pathologist’s knowledge of tissue and cell disorders. In the face of these problems, many pathologists feel that the necessary investment in time and money is not worthwhile. In order to bridge the gap between conventional practice in pathology and sophisticated cytometry and histometry technology, the feasibility of direct interfacing between microscope and computer has been studied in the framework of the European research and development action AIM (Advanced Informatics in Medicine). This project named HOME: Highly Optimized Microscope Environment [2] is an interlaboratory coordinated effort to introduce computer assistance in pathology practice. The overall project involves in depth analysis of i) medical motivations and requirements, ii) elligibility of cell and tissue measurements, and iii) current practice at both the laboratory and the medical personnel levels. This paper presents the hardware and software prototype designed for some simple functions identified as basic in computer assisted pathology.
MATERIALS AND METHODS HOME is a user-friendly computerized microscope which looks like a conventional microscope with a mouse attached. It is designed specifically to provide the pathologist with simple facilities including the combination of microscope, display, and stage to be used for locating and relocating individual cells in pathological specimens (i.e., cervical smears); the simple morphological measurements (such as length) on typical histological specimens (i.e., muscle fibres); and the use of menu control within the microscope field to
allow computer-innocent users to carry out analytical functions, and some of the ancillary functions related to the management of specimens in a routine pathology laboratory environment. These facilities make it possible to use a computer without having to look up from the microscope. They are obtained by combining a range of computerized devices with the conventional light microscope. The first is a computer display whose image is superimposed on the optical field image of the microscope. This allows the user to see computer-generated texts and graphics in the microscope image. Also, in conjunction with a mouse, the computer display enables the user to control a cursor which can “draw on” the microscope image and can be used to control computer software through menus and light buttons. This principle is not new [3,6,81, but is made more practical by the availability of small, high-brightness display tubes primarily developed for aviation technology and dedicated software. The second is a standardised computer-readable slide coordinate system based on high accuracy transducers fixed to the conventional microscope stage. The third is a computer-readable objective turret encoding system. The basic question may be raised whether it is more pragmatic to project the computer monitor text and graphics onto the optical microscope image or display both microscope image and computer text and graphics on a high quality high resolution color monitor. The rationale for the technological choice which has been made is i) the pathologists will not make a decision on abnormalities on the basis of a video image alone of the specimen, even of high quality; ii) the optical microscope image, although rather flat, is actually three dimensional and basically more informative than the two-dimensional video image; iii) the neck and back strain due to staring through the microscope is not worse than the visual fatigue due to scrutinizing at a video monitor; and iv) whatever the improvements of the video camera and monitor may ultimately be, the overall quality of the resulting electronic image cannot compete with the resolution of the optical image, particularly for small details of diagnostic relevance.
HARDWARE System Overview A block diagram of the basic HOME prototype system hardware is shown in Figure 1. The computer board is IBM-PC-compatible. For development purposes, a COMPAQ 80 386 computer running MS Windows under the MS-DOS operating system was used. The principle of the optical image mixing for the HOME microscope is shown in Figure 2. The two component images are passed through a half-silvered mirror and the combined image is viewed through the normal microscope eyepieces. The HOME prototype, presented in Figure 3, has been designed for two commercially available microscope models, the DIAPLAN (Leitz Wetzlar, Germany) and the AXIOSCOP 20 (Carl Zeiss, Oberkochen, Germany) from manufacturers col-
HIGHLY OPTIMIZED MICROSCOPE ENVIRONMENT
1-
1
VIDEOINPUT
OBJECTIVE ENCODER
111
=
STAGE ENCODER
I
MICROSCOPE
COMPUTER
I
FIG. 1. Diagram of the basic HOME prototype system. The microscope is turned into a computer workstation only driven by means of a mouse. The position of the stage and the magnification of the objective are encoded and transmitted to the computer central unit in real time. The computer display is projected into the microscope image using the optical beam mixing shown in Figure 2.
VIDEO
OUTPUT
4 ,
4 ,
u
VIDEO INPUT # -
A
Half silvered mirror EYEPIECES
Objective Specimen Light Source
FIG. 2. Principal of the optical image mixing for the HOME microscope. The image from the computer display (video input) and the image of the specimen are both passed through a half-silvered mirror so that the combined image is viewed through the normal microscope eyepieces. A video output is provided for image capture when the HOME microscope is used for interfacing image processing and analysis.
FIG. 3. HOME prototypes designed for (A) the DIAPLAN (Leitz Wetzlar, Germany) and (B) the AXIOSCOP 20 (Zeiss, Oberkochen, Germany) microscopes. The Leitz unit is based on the Microvid system, while the Zeiss unit was specially designed for this project.
laborating on the HOME project. In both cases the microscopes were modified by the manufacturers to provide the display facility. The Leitz unit consists of a commercially available Microvid system, while the Zeiss unit was specially designed for this project and is described more specifically below.
driver cards. The displayed texts and graphics are green and thus offer a maximum contrast with the usual cell and tissue staining. In both the Zeiss and Leitz prototypes, the video display is driven by a HERCULES standard graphics board which gives a monochrome, single-brightness display of 720 points by 350 lines. An MS-DOS switch routine was written to divert all screen output from the normal VGA computer display to the HERCULES display.
Microscope Video-Head The video overlay head consists of a beam-splitting prismimirror with a transmittance to reflectance ratio of 70130. An optical switch allows the user to select either the optical image alone, or the mixed video plus optical image. A separate output port is provided for TV camera either built-in and/or external. The video monitor is a small (1”) high-brightness monochrome tube mounted in the microscope video-head with its
Encoding Stage The method of implementation chosen for the HOME prototypes is the manual encoding stage, in which the conventional microscope stage (manually driven using the normal X and Y knobs} was equipped with linear position encoders which enable the computer to read the current position of the stage at any time. The prototype encoding stages shown in Figure 4 were built by Apollo Optical Services Ltd (Camberley, UK). The lin-
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FIG.4. Prototype encoding stage showing one of the Sony Magnescale fitted by Apollo Optical Services, Ltd. (Camberley, UK).
ear position encoders are the “Skeleton” scales (Sony Magnescale) fitted to the X and Y stage axes, respectively, and connected to readout units (Sony LH20). The output was passed to the computer via a single RS 232C (4800 baud) serial line.
Slide Clip A slide clip, shown in Figure 5, has been specially designed [41 to provide accurate and reproducible positionning of slides on the stage, and accurate rotational alignment of the coordinate system axes. To ensure that this occurs with a minimum of operator assistance the two sprung arms are operated by a single lever and push the slide sequentially against the stops. The rotation adjustment necessary to give accurate rotational alignment between the stage axis and the slide axis is provided by two locking screws in slotted holes, together with a central bearing screw. A graticule slide must be used to set the alignment and to define a fixed origin position for the coordinate system. Objective Turret Encoder The Zeiss prototype includes a standard magnetic objective turret encoder that enables the computer system to automatically adapt the display scale to the objective in use and to record, whenever useful, which objective has been used and where during the observation of a specimen [31. SOFTWARE All software for the HOME prototype was written in the C language, to run under the MS-DOS/MSWindows 3 environment. The software is structured as a series of modules, which can be combined to form application programs designed to suit particular applications. Some of the facilities and procedures available with the present software are described below.
FIG. 5. Slide clip specially designed for the HOME microscope. The two sprung arms are operated by a single lever and push the slide sequentially against the stops to provide accurate and reproducible positionning of slides on the stage. The rotation adjustment necessary to give accurate rotational alignment between the stage axis and the slide axis is provided by two locking screws in slotted holes, together with a central bearing screw.
Menu Control It is intended that all user interaction with HOME software should be carried out through menu displays in the microscope field, together with movement of a mouse controlled cursor and operation of the mouse buttons. In the prototype systems, a two-button mouse is used; most operations can be carried out with single or double clicks on the main (left) button, but doubleclick operations are copied on the right buttons to suit the preferences of the operator. Note that the conventional computer keyboard is not used by HOME software. As shown in Figure 3, the mouse is the only visible device added to the microscope which is relayed to the computer housed elsewhere. Coordinate Calibration and Setup The coordinate system for the HOME prototypes was defined using the England Finder graticule slide (Graticules, Ltd, Tunbridge Wells, UK) as a reference. This slide contains a n evaporated metalised pattern of 1mm squares, with numbers and letters, accurately placed with respect to marked slide edges. The origin of the coordinate system is defined a s the centre of a selected fiducial mark using the 20 x objective. All absolute coordinate measurements are then automatically converted into micrometers from this origin point whichever the objective in use. The coordinate system setup software incorporates a series of diagrammatic menu displays for zeroing the linear encoder readings at the appropriate position on the England Finder (this must be done whenever the stage encoders are switched off), measuring the exact magnification factors and centrality offsets for each of the objective lenses on the microscope (this must be done whenever the objectives are
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ilar to that used for user identifier input (Fig. 6). Second, if a file for the specimen already exists on the system, then the number can be loaded directly by clicking the appropriate entry in a scrolled file list displayed with the keypad. This facility would be useful for checking or re-analysing specimens for which HOME results already exist, or when the HOME system is networked t o a separate data entry station which generates request lists.
Object Marking This procedure enables the user to flag objects in the microscope field such as cells, fibres etc. by clicking on the object centre, after which an icon is shown at the appropriate point on the display. A menu of different icons can be displayed to allow the user to differentiate objects of various types as shown in Figure 7. The HOME system software moves displayed icons to follow the parent object whenever the stage is moved, and also when a different microscope objective lens is used. Displayed object markers can be deleted either by doubleclicking the left-hand button or by a single click with the right-hand mouse button.
FIG.6. Entering textual information into the HOME files is done by pointing at the displayed numbers or messages with the mousecontrolled cursor arrow. Identification of the user or the slide (A), as well as the conclusions of the examination of the specimen (B)are thus entered in standardized format and sequence.
removed andlor replaced), and adjusting the rotation of the slide clip to ensure that a defined line on the England Finder is exactly parallel with the X stage axis (this must be done whenever the microscope stage is moved or replaced).
User Identifier Number Input This procedure can be used to limit access t o medical and other information in the HOME system to accredited personnel. The user must give a correct personal User Identifier Number to the system before he is allowed to enter the HOME software and medical files. This is supplied by pointing at numbers on a displayed keypad menu with the mouse-controlled cursor arrow as shown in Figure 6.
Slide Reference Number Input This procedure allows the input of a Slide Reference Number to the system for use in reporting, filing, etc. of results. Two methods have been provided in the prototype system software. Firstly, the number can be input manually using a mouse-driven keyboard display sim-
Object Relocation The position of microscope fields containing any marked object on the current specimen can be displayed on a small diagram of the slide. This slide icon also shows a “current position” marker, which moves as the stage is moved. With this facility, illustrated in Figure 7, a marked object can be quickly relocated by moving the stage until the “current position” marker is aligned with its position marker. The required object, with its marker, will then enter the microscope field.
Length Measurements This procedure is a simple example of a morphometric feature measurement. It allows the user to measure any length (such as muscle-fibre diameter) by singleclicking at the required start point, and then moving the cursor to the end point, and single-clicking. The displayed chord then collapses to a central marker icon, with a value and coordinate position (this prevents remeasurement of an already-measured object). As above, markers move with their parent object and can be deleted.
Information Display The type of display can be used to enable the operator to call up items of information which may be required during the analysis of a specimen, without having to look up from the microscope. Typical information is patient name, sex, and age; counts, means, and statistical confidence information of measurements so far made; selected objective lens, stage position etc. Also, histograms and scatter diagrams can be displayed t o show details of the measurements so far made on the current specimen. Moreover, the observer can input information by simply clicking on the appropriate dia-
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to printifile this information for quality control purposes for tasks such a s cervical smear screening.
RESULTS AND DISCUSSION Initial experience with both the Leitz MICROVID and Zeiss HOME prototype systems indicates that the addition of the computer display to the light microscope provides a convenient and easy-to-use human-computer interface. I n both cases, the display tube and beam-mixing optics together provide a computer display that is easy to see and which does not disturb the conventional observation of the specimen. The mouse allows the user to position precisely the cursor “in” the microscope image, and this. together with suitable menu structures and real time control of stage position and objective magnification, has proved a user-friendly way of transforming the microscope into a dedicated computer workstation compatible with daily routine tasks in the pathology laboratory.
FIG.7. Examples of the HOME icons to flag cells whose type can be specified by a particular mark (A) to control which part of the slide has been actually observed at any time (B), t o see where the flagged objects are located within the slide (C rectangular marks), and to know the current position of the slide and track its motion (C round mark moving to the right). The magnification of the objective in use is displayed at the top right corner.
logue box whose message is standardised for a given application (Fig. 6).
Stage Tracking Using the stage icon as described above, the track of the stage can be shown during the scan of a specimen slide. In this way, illustrated in Figure 7, it is possible for the operator to see how much, and which parts, of the slide have so far been examined. It is also possible
User-Friendliness Five HOME prototypes have so far been constructed, three based on the Zeiss AXIOSCOP 20 and two with the Leitz MICROVID system. To date, these have been used mostly for software and hardware development. Recently, however, one system has been installed in St. Bartholomew’s Hospital (Professor G. Slavin, London, UK) for evaluation purposes. Also, limited operating experience has been gained by the other pathologists in the HOME consortium, and by others during the 12th Meeting of the Pathological Society of Great Britain and Ireland (January 1991). It has been found that most personnel, after a few minutes experience, are able to use mouselcursorimenu displays to control program options and input numbers with the “key-pad” facility, and also to make accurate cursor positioning for morphometric measurements. I n general, pathologists and technical staff who have had previous experience with other morphometric or image analysis systems felt that the HOME system appeared to be definitely more user-friendly for routine laboratory use. Initially, the system is to be used for two specific applications, cervical smear screeningireviewing and for morphological measurements on muscle fibre specimens [ l l l . Relocation Accuracy The encoding stage and encoding objective turret greatly enhances the usefulness of the HOME system. Of particular value is the ability to mark and relocate cells/objects independently of stage position, so that registration between computer markers and the microscopic object is maintained when the objective lens is changed, when the stage is moved, or even when the slide is loaded on another HOME microscope. To determine the accuracy of the encoding stage system, several measurements were made with 10 x and 40 x objectives, using a small object on a conventional microscope slide, together with the England Finder slide
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Table 1 Error Components (in pm) of the Coardinate Measurement Using a Microscope Encoding Stage Equipped With Sony Transducers on the X and Y Axes and a England Finder Reference Slide x 10
x 40
X Y X Y Objective Sony encoder repeatability 1.0 1.0 1.0 1.0 2.9 1.3 2.9 1.3 Mean stage backlash 0.6 0.9 2.6 3.6 Max. display pixel error 0.6 2.2 0.6 Slide clip reload error (s.d) 2.2 Objective turret reload backlash 8.4 0 2.1 0 Sony encoder error 2.75 2.75 2.75 2.75 5 25 5 Max. England Finder error 25 6.7 20 Max, stage rotation setup error 6.7 20
for coordinate setup where appropriate. The results obtained in these tests are shown in Table 1. From this data, error distributions of object positions under various conditions can be computed as in Table 2. These computations suggest that the basic aim of unique registration of a typical cell nucleus (which is around 10 km in diameter) can be achieved on the prototype HOME systems when the location and subsequent relocation are carried out on the same microscope, with coordinate setup carried out with the same England Finder graticule slide (25.3 pm error with 40 x objective). However, larger errors ( k 3 7 km) may occur in cross-registration between different microscopes, due to differences in slide-clip rotation and variation in England Finder graticule slides. In practice, these errors are unlikely to prevent correct cell relocation in most practical situations because they are systematic, and the user will quickly adapt to their effect. However, user-controlled offset facilities and improved position encoding system are under development to increase the accuracy further.
Length Measurement Accuracy A test was carried out on a prototype HOME system to estimate the accuracy of the length measurement facility. The longest chord of a typical large muscle fibre was measured repeatedly (by a single operator) with different objective magnifications, and the standard errors of each set of measurements estimated. The results are shown in Table 3. At the centre of the field, the measurement means are very similar at all magnifications, but accuracy obviously increases as higherpower objectives are used. However, when the same object is measured at a far corner of the field, the measurements are substantially different. This error is due to differential distortion between the microscope and display images. Methods of software correction for this distortion are now being investigated. Potential Applications The basic HOME system is potentially useful in a wide range of applications [ll]. First, there are many applications for object marking facilities, either for ex-
Table 2 Overall Relocation Accuracy (in pm) Using a Microscope Encoding Stage Equipped With Sony Transducers on the X and Y Axes and a England Finder Reference Slide x 40
x 10
Obiective Without reload With reload (same microscope, no re-zero) With reload (same microscope, turret turned) With reload (same microscope, with re-zero) With reload
(another microscope)
X
Y
X
Y
4.0
4.0
2.1
1.9
4.6
4.0
3.8
2.0
9.6
4.0
4.4
2.0
8.5
5.5
5.3
2.6
37.3 30.0 37.1 29.7
Table 3 Length Measurement Accuracy (in pm) Using a Microscope Encoding Stage Equipped With Sony Transducers on the X and Y Axes Centre of the field Objective x 10 x 20 x 40
(n = 10) Mean Sd. 142.2 1.18 0.83 140.5 142.8 0.73
Top right corner (n = 10) Mean Sd. 131.9 132.6 134.0
2.36 0.78 0.49
amination by other personnel (e.g., regions of abnormality in tissue sections, cervical smear screening, and marking of cytogenetic abnormalities), or for recordkeeping purposes. Second, the system can be used to carry out morphometric measurements, either in general-purpose pathology use or for specific morphometric tissue-specific procedures (muscle, bone, and gut). Third, the system may be used for counting tasks (differential cell counts, mitotic index, and pycnotic index). Fourth, by displaying suitable grid patterns stereology applications can be implemented. Last, the system may be used for teaching purposes, either by displaying good examples of a particular cell type in a specimen, or for more advanced computer-aided instruction techniques. The addition of other facilities would allow the HOME system to be used for a n even wider range of applications. First, a motorized stage would permit much faster relocation of objects, which would be extremely valuable in some applications such a s cervical cytology screening. Second, the addition of a video camera would facilitate remote decision-sharing on difficult specimens or cells. Third, the provision of image analysis would greatly extend the range of measurements possible on manually selected cells or regions (in particular, DNA-ploi'dy and receptors) and would allow the introduction of fully automatic analysis of suitable objects in selected fields. Fourthly, networking facilities connecting several HOME workstations and data entry workstations, HOME automatic slide scanners equipped with motorized stage and image analysis facilities, and hospital information networks will greatly
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improve the information transfer in the routine pathology laboratory. Last, the addition of flexible self-proGarnming facilities would allow the pathologist to set up and test new measurement procedures and to export particularly successful procedures for general use.
ACKNOWLEDGMENTS The a u t h o r s w i s h t o express their indebtedness t o the p a thologist members of the HOME consortium: Pr. Etienne Martin (Bicstre, France); D r . A n n i e Morens (Grenoble, F r a n c e ) ; Pr. Peter Pfitzer (Diisseldorf, Germany); Pr.F r a n c o Rilke (Milano, Italy); Pr. Gerard Slavin (London, United Kingdom), and Pr. Peter Vooijs (Nijmegen, Netherlands); w i t h o u t their h e l p the project would n o t h a v e been possible. The a u t h o r s also w i s h t o express their grateful thanks t o individual m e m b e r s of the industrial p a r t n e r s in the project, in p a r t i c u l a r Dr. Nasse, Dr. Kohlhase, and Dr. Gausman of Carl Zeiss Gmbh, and D r . P. den Englese of Leitz Microscan, for their h e l p in designing the optical s y s t e m s for this project and also to Mr. P. Riley of Apollo Optical Services for his help in the design of the encoding stage and stage clip. The skillful assistance of Michele Brugal for documentation, Yolande B o u v a t for iconography, and Victoria von Hagen for m a n u s c r i p t preparation is acknowledged.
LITERATURE CITED 1. Baak JPA: Quantitative Pathology today: A technical review. Pathol Res Pract 182:396-400, 1987. 2. Brugal G. Pattern recognition, image processing, related data
analysis and expert systems integrated in medical microscopy. In: Proc. 9th Int. Conf. on Pattern Recognition, Roma (Italy). IEEE Computer Society Press, 1988, pp. 286-293. 3. Christiaanse JGM, Koper GJM, Verwoerd NP, Bonnet J , Ploem JS: A microscope for off-line relocation of LEYTAS detected objects. In: Abstracts of Analytical Cytology and Cytometry IX, Schloss Elmau, Germany, 1982, p. 42. 4. Dye R, Farrow ASJ, Fletcher DS, Tucker JH: Microscope Slide Clip. Patent Application No. 9019979.5, 1990. 5. Franklin WA, Bibbo M, Doria MI, Dytch HE, Toth J, DeSombre E, Wied GL: Quantitation of Estrogen receptor content and Ki-67 staining in breast carcinoma by the MicroTICAS image analysis system. Analyt Quant Cytol Histom 9:279-286, 1987. 6. Glaser EM, Van der Loos H: Computer Microscope Apparatus and method for superimposing an electronically produced image from the computer memory upon the image in the microscope field. US Patent 4.202.037. US Patent Ofice, Washington DC. 7. Gundersen HJG, Bendtsen TF, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sorensen FB, Vesterby A, West MJ: Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96: 379-394, 1988. 8. Schmitt P: Leitz MICROVID Modul zur Computer-Mikrospie. Ernst Leitz Wetzlar GMBH. 9. Seckinger D, Sugarbaker E, Frankfurt 0: DNA Content in Human Cancer. Arch Pathol Lab Med 1 1 3 5 1 9 4 2 6 , 1989. 10. Silverman JF, Finley JL, OBrien KF, Dabbs DJ, Park HK, Larkin EW: Diagnostic accuracy and role of immediate interpretation of fine needle aspiration biopsy specimens from various sites. A d a Cytol 33:791-796, 1989. 11. Von Hagen V, Morens A, Krief B: Highly optimized microscope environment: A new workstation for microscopic analysis in patholom. Anal Cell Pathol 3:249-254. 1991. 0”
0 1992 Wiley-Liss, Inc.
HOME: Highly Optimized Microscope Environment’ Gerard Brugal, Richard Dye, Bruno Krief, Jean-Marc Chassery, Hans Tanke, and J a m e s H. Tucker Equipe de Reconnaissance des Formes et Microscopie Quantitative, CERMO, Grenoble, France (G.B., B.K., J.-M.C.); Medical Research Council Human Genetics Unit, Edinburgh, Scotland, United Kingdom (R.D., J.H.T.); and Sylvius Laboratories, University of Leiden, Leiden, Netherlands (H.T.) Received for publication April 15, 1991; accepted July 31, 1991
The Highly Optimized Microscope Environment (HOME) is a computerized microscope designed to assist pathologists and cytotechnicians in clinical routine tasks. The prototype system consists of a IBM-PC compatible computer and a light microscope in which a built-in highresolution computer display image is superimposed on the optical image of the specimen. Also, a manually operated encoding stage and objective turret encoder are used to provide continuous monitoring of the stage coordinates and microscope magnification to the computer. This allows any position on a slide to be uniquely defined and makes it possible to measure interactively lengths and areas larger than the size of the microscope field. Software, written in the C language and operating under the MS-DOSMS-Windows environment, is
In modern hospitals, the pathology laboratory plays a n essential role in the diagnosis and prognosis of most serious diseases, and therefore in the subsequent medical decision-making and health care for the patient. In spite of this essential role, pathology practice has to date remained relatively untouched by recent developments in information technology: routine pathology is still carried out largely by microscopic examination of specimens by the pathologist, followed by the reporting of his opinion concerning the appearance of the specimen back to the requesting clinician. While this is satisfactory for the majority of specimens, for most types of pathological sample there is a “grey area” containing some specimens for which the result is not clear and is therefore subjective, unreliable, and poorly reproducible [lo]. One approach to improving the diagnosis for such specimens is the introduction of quantitative analysis. Counting is the first quantitative approach which has been used extensively in haematology (differential
controlled by means of a mouse-driven cursor moving over menu light-buttons displayed on the microscope image. The HOME microscope workstation is potentially useful in a wide range of applications such as i) tagging information on particular cells and tissue structures that can thus be accurately located and relocated, ii) performing morphometric measurement, differential counting, and stereological assessment of biological specimens, and iii) training and educating laboratory personnel. Finally, HOME will offer in the near future a userfriendly interface for automatic image processing of cells and tissue entities in interactively selected specimen areas. Key terms: Microscopy, image cytometry, image histometry, computerized microscopy
white blood cell count), tumour pathology (mitotic index), and hormonal cytology (pycnotic index). Several other quantitative techniques for pathology have been investigated extensively for many years. Interactive computer morphometry, in which the user can delineate lengths and areas on a projected microscope image using a digital tablet or similar instrument, has been found to be diagnostically useful in many types of tumour pathology, bone pathology, and muscle pathology 111. Stereology, the use of statistical sampling to estimate geometrical properties of specimens, has also been applied to similar problems [7]. Moreover, densitometry has been very widely used to measure total
‘The project was partly funded by the Commission of the European Communities under its Advanced Informatics in Medicine (AIM) action, Contract A 1007.
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cellular DNA content and other cell constituents [9], and more recently for the color analysis of a variety of immunocytochemical markers [5]. All these measurements can be carried out using image cytometry (either interactive or automatic). However, in spite of these achievements there has been a disappointing impact on pathology practice. These techniques so far have been confined mainly to research laboratories, and are still not used widely in routine pathology. A major reason for this is lack of suitable equipment. Although a number of general-purpose computer morphometry and image analysis systems are commercially available, these are often large and expensive, and require substantial expertise to reconfigure for the pathologist requirements. Also, they are not satisfactory for measurement on complex biological specimens: often the user must oscillate between microscope and computer, first using the microscope and its control to find a suitable field to measure and then having to tu rn to the computer keyboard and screen to carry out the required measurement operations. Usually, this cycle must be repeated many times to obtain a statistically representative value for the overall specimen. For this reason the acquisition of diagnostically relevant information with these systems is often labour-intensive, time-consuming, and poorly correlated with the conventional pathologist’s knowledge of tissue and cell disorders. In the face of these problems, many pathologists feel that the necessary investment in time and money is not worthwhile. In order to bridge the gap between conventional practice in pathology and sophisticated cytometry and histometry technology, the feasibility of direct interfacing between microscope and computer has been studied in the framework of the European research and development action AIM (Advanced Informatics in Medicine). This project named HOME: Highly Optimized Microscope Environment [2] is an interlaboratory coordinated effort to introduce computer assistance in pathology practice. The overall project involves in depth analysis of i) medical motivations and requirements, ii) elligibility of cell and tissue measurements, and iii) current practice at both the laboratory and the medical personnel levels. This paper presents the hardware and software prototype designed for some simple functions identified as basic in computer assisted pathology.
MATERIALS AND METHODS HOME is a user-friendly computerized microscope which looks like a conventional microscope with a mouse attached. It is designed specifically to provide the pathologist with simple facilities including the combination of microscope, display, and stage to be used for locating and relocating individual cells in pathological specimens (i.e., cervical smears); the simple morphological measurements (such as length) on typical histological specimens (i.e., muscle fibres); and the use of menu control within the microscope field to
allow computer-innocent users to carry out analytical functions, and some of the ancillary functions related to the management of specimens in a routine pathology laboratory environment. These facilities make it possible to use a computer without having to look up from the microscope. They are obtained by combining a range of computerized devices with the conventional light microscope. The first is a computer display whose image is superimposed on the optical field image of the microscope. This allows the user to see computer-generated texts and graphics in the microscope image. Also, in conjunction with a mouse, the computer display enables the user to control a cursor which can “draw on” the microscope image and can be used to control computer software through menus and light buttons. This principle is not new [3,6,81, but is made more practical by the availability of small, high-brightness display tubes primarily developed for aviation technology and dedicated software. The second is a standardised computer-readable slide coordinate system based on high accuracy transducers fixed to the conventional microscope stage. The third is a computer-readable objective turret encoding system. The basic question may be raised whether it is more pragmatic to project the computer monitor text and graphics onto the optical microscope image or display both microscope image and computer text and graphics on a high quality high resolution color monitor. The rationale for the technological choice which has been made is i) the pathologists will not make a decision on abnormalities on the basis of a video image alone of the specimen, even of high quality; ii) the optical microscope image, although rather flat, is actually three dimensional and basically more informative than the two-dimensional video image; iii) the neck and back strain due to staring through the microscope is not worse than the visual fatigue due to scrutinizing at a video monitor; and iv) whatever the improvements of the video camera and monitor may ultimately be, the overall quality of the resulting electronic image cannot compete with the resolution of the optical image, particularly for small details of diagnostic relevance.
HARDWARE System Overview A block diagram of the basic HOME prototype system hardware is shown in Figure 1. The computer board is IBM-PC-compatible. For development purposes, a COMPAQ 80 386 computer running MS Windows under the MS-DOS operating system was used. The principle of the optical image mixing for the HOME microscope is shown in Figure 2. The two component images are passed through a half-silvered mirror and the combined image is viewed through the normal microscope eyepieces. The HOME prototype, presented in Figure 3, has been designed for two commercially available microscope models, the DIAPLAN (Leitz Wetzlar, Germany) and the AXIOSCOP 20 (Carl Zeiss, Oberkochen, Germany) from manufacturers col-
HIGHLY OPTIMIZED MICROSCOPE ENVIRONMENT
1-
1
VIDEOINPUT
OBJECTIVE ENCODER
111
=
STAGE ENCODER
I
MICROSCOPE
COMPUTER
I
FIG. 1. Diagram of the basic HOME prototype system. The microscope is turned into a computer workstation only driven by means of a mouse. The position of the stage and the magnification of the objective are encoded and transmitted to the computer central unit in real time. The computer display is projected into the microscope image using the optical beam mixing shown in Figure 2.
VIDEO
OUTPUT
4 ,
4 ,
u
VIDEO INPUT # -
A
Half silvered mirror EYEPIECES
Objective Specimen Light Source
FIG. 2. Principal of the optical image mixing for the HOME microscope. The image from the computer display (video input) and the image of the specimen are both passed through a half-silvered mirror so that the combined image is viewed through the normal microscope eyepieces. A video output is provided for image capture when the HOME microscope is used for interfacing image processing and analysis.
FIG. 3. HOME prototypes designed for (A) the DIAPLAN (Leitz Wetzlar, Germany) and (B) the AXIOSCOP 20 (Zeiss, Oberkochen, Germany) microscopes. The Leitz unit is based on the Microvid system, while the Zeiss unit was specially designed for this project.
laborating on the HOME project. In both cases the microscopes were modified by the manufacturers to provide the display facility. The Leitz unit consists of a commercially available Microvid system, while the Zeiss unit was specially designed for this project and is described more specifically below.
driver cards. The displayed texts and graphics are green and thus offer a maximum contrast with the usual cell and tissue staining. In both the Zeiss and Leitz prototypes, the video display is driven by a HERCULES standard graphics board which gives a monochrome, single-brightness display of 720 points by 350 lines. An MS-DOS switch routine was written to divert all screen output from the normal VGA computer display to the HERCULES display.
Microscope Video-Head The video overlay head consists of a beam-splitting prismimirror with a transmittance to reflectance ratio of 70130. An optical switch allows the user to select either the optical image alone, or the mixed video plus optical image. A separate output port is provided for TV camera either built-in and/or external. The video monitor is a small (1”) high-brightness monochrome tube mounted in the microscope video-head with its
Encoding Stage The method of implementation chosen for the HOME prototypes is the manual encoding stage, in which the conventional microscope stage (manually driven using the normal X and Y knobs} was equipped with linear position encoders which enable the computer to read the current position of the stage at any time. The prototype encoding stages shown in Figure 4 were built by Apollo Optical Services Ltd (Camberley, UK). The lin-
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FIG.4. Prototype encoding stage showing one of the Sony Magnescale fitted by Apollo Optical Services, Ltd. (Camberley, UK).
ear position encoders are the “Skeleton” scales (Sony Magnescale) fitted to the X and Y stage axes, respectively, and connected to readout units (Sony LH20). The output was passed to the computer via a single RS 232C (4800 baud) serial line.
Slide Clip A slide clip, shown in Figure 5, has been specially designed [41 to provide accurate and reproducible positionning of slides on the stage, and accurate rotational alignment of the coordinate system axes. To ensure that this occurs with a minimum of operator assistance the two sprung arms are operated by a single lever and push the slide sequentially against the stops. The rotation adjustment necessary to give accurate rotational alignment between the stage axis and the slide axis is provided by two locking screws in slotted holes, together with a central bearing screw. A graticule slide must be used to set the alignment and to define a fixed origin position for the coordinate system. Objective Turret Encoder The Zeiss prototype includes a standard magnetic objective turret encoder that enables the computer system to automatically adapt the display scale to the objective in use and to record, whenever useful, which objective has been used and where during the observation of a specimen [31. SOFTWARE All software for the HOME prototype was written in the C language, to run under the MS-DOS/MSWindows 3 environment. The software is structured as a series of modules, which can be combined to form application programs designed to suit particular applications. Some of the facilities and procedures available with the present software are described below.
FIG. 5. Slide clip specially designed for the HOME microscope. The two sprung arms are operated by a single lever and push the slide sequentially against the stops to provide accurate and reproducible positionning of slides on the stage. The rotation adjustment necessary to give accurate rotational alignment between the stage axis and the slide axis is provided by two locking screws in slotted holes, together with a central bearing screw.
Menu Control It is intended that all user interaction with HOME software should be carried out through menu displays in the microscope field, together with movement of a mouse controlled cursor and operation of the mouse buttons. In the prototype systems, a two-button mouse is used; most operations can be carried out with single or double clicks on the main (left) button, but doubleclick operations are copied on the right buttons to suit the preferences of the operator. Note that the conventional computer keyboard is not used by HOME software. As shown in Figure 3, the mouse is the only visible device added to the microscope which is relayed to the computer housed elsewhere. Coordinate Calibration and Setup The coordinate system for the HOME prototypes was defined using the England Finder graticule slide (Graticules, Ltd, Tunbridge Wells, UK) as a reference. This slide contains a n evaporated metalised pattern of 1mm squares, with numbers and letters, accurately placed with respect to marked slide edges. The origin of the coordinate system is defined a s the centre of a selected fiducial mark using the 20 x objective. All absolute coordinate measurements are then automatically converted into micrometers from this origin point whichever the objective in use. The coordinate system setup software incorporates a series of diagrammatic menu displays for zeroing the linear encoder readings at the appropriate position on the England Finder (this must be done whenever the stage encoders are switched off), measuring the exact magnification factors and centrality offsets for each of the objective lenses on the microscope (this must be done whenever the objectives are
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113
ilar to that used for user identifier input (Fig. 6). Second, if a file for the specimen already exists on the system, then the number can be loaded directly by clicking the appropriate entry in a scrolled file list displayed with the keypad. This facility would be useful for checking or re-analysing specimens for which HOME results already exist, or when the HOME system is networked t o a separate data entry station which generates request lists.
Object Marking This procedure enables the user to flag objects in the microscope field such as cells, fibres etc. by clicking on the object centre, after which an icon is shown at the appropriate point on the display. A menu of different icons can be displayed to allow the user to differentiate objects of various types as shown in Figure 7. The HOME system software moves displayed icons to follow the parent object whenever the stage is moved, and also when a different microscope objective lens is used. Displayed object markers can be deleted either by doubleclicking the left-hand button or by a single click with the right-hand mouse button.
FIG.6. Entering textual information into the HOME files is done by pointing at the displayed numbers or messages with the mousecontrolled cursor arrow. Identification of the user or the slide (A), as well as the conclusions of the examination of the specimen (B)are thus entered in standardized format and sequence.
removed andlor replaced), and adjusting the rotation of the slide clip to ensure that a defined line on the England Finder is exactly parallel with the X stage axis (this must be done whenever the microscope stage is moved or replaced).
User Identifier Number Input This procedure can be used to limit access t o medical and other information in the HOME system to accredited personnel. The user must give a correct personal User Identifier Number to the system before he is allowed to enter the HOME software and medical files. This is supplied by pointing at numbers on a displayed keypad menu with the mouse-controlled cursor arrow as shown in Figure 6.
Slide Reference Number Input This procedure allows the input of a Slide Reference Number to the system for use in reporting, filing, etc. of results. Two methods have been provided in the prototype system software. Firstly, the number can be input manually using a mouse-driven keyboard display sim-
Object Relocation The position of microscope fields containing any marked object on the current specimen can be displayed on a small diagram of the slide. This slide icon also shows a “current position” marker, which moves as the stage is moved. With this facility, illustrated in Figure 7, a marked object can be quickly relocated by moving the stage until the “current position” marker is aligned with its position marker. The required object, with its marker, will then enter the microscope field.
Length Measurements This procedure is a simple example of a morphometric feature measurement. It allows the user to measure any length (such as muscle-fibre diameter) by singleclicking at the required start point, and then moving the cursor to the end point, and single-clicking. The displayed chord then collapses to a central marker icon, with a value and coordinate position (this prevents remeasurement of an already-measured object). As above, markers move with their parent object and can be deleted.
Information Display The type of display can be used to enable the operator to call up items of information which may be required during the analysis of a specimen, without having to look up from the microscope. Typical information is patient name, sex, and age; counts, means, and statistical confidence information of measurements so far made; selected objective lens, stage position etc. Also, histograms and scatter diagrams can be displayed t o show details of the measurements so far made on the current specimen. Moreover, the observer can input information by simply clicking on the appropriate dia-
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to printifile this information for quality control purposes for tasks such a s cervical smear screening.
RESULTS AND DISCUSSION Initial experience with both the Leitz MICROVID and Zeiss HOME prototype systems indicates that the addition of the computer display to the light microscope provides a convenient and easy-to-use human-computer interface. I n both cases, the display tube and beam-mixing optics together provide a computer display that is easy to see and which does not disturb the conventional observation of the specimen. The mouse allows the user to position precisely the cursor “in” the microscope image, and this. together with suitable menu structures and real time control of stage position and objective magnification, has proved a user-friendly way of transforming the microscope into a dedicated computer workstation compatible with daily routine tasks in the pathology laboratory.
FIG.7. Examples of the HOME icons to flag cells whose type can be specified by a particular mark (A) to control which part of the slide has been actually observed at any time (B), t o see where the flagged objects are located within the slide (C rectangular marks), and to know the current position of the slide and track its motion (C round mark moving to the right). The magnification of the objective in use is displayed at the top right corner.
logue box whose message is standardised for a given application (Fig. 6).
Stage Tracking Using the stage icon as described above, the track of the stage can be shown during the scan of a specimen slide. In this way, illustrated in Figure 7, it is possible for the operator to see how much, and which parts, of the slide have so far been examined. It is also possible
User-Friendliness Five HOME prototypes have so far been constructed, three based on the Zeiss AXIOSCOP 20 and two with the Leitz MICROVID system. To date, these have been used mostly for software and hardware development. Recently, however, one system has been installed in St. Bartholomew’s Hospital (Professor G. Slavin, London, UK) for evaluation purposes. Also, limited operating experience has been gained by the other pathologists in the HOME consortium, and by others during the 12th Meeting of the Pathological Society of Great Britain and Ireland (January 1991). It has been found that most personnel, after a few minutes experience, are able to use mouselcursorimenu displays to control program options and input numbers with the “key-pad” facility, and also to make accurate cursor positioning for morphometric measurements. I n general, pathologists and technical staff who have had previous experience with other morphometric or image analysis systems felt that the HOME system appeared to be definitely more user-friendly for routine laboratory use. Initially, the system is to be used for two specific applications, cervical smear screeningireviewing and for morphological measurements on muscle fibre specimens [ l l l . Relocation Accuracy The encoding stage and encoding objective turret greatly enhances the usefulness of the HOME system. Of particular value is the ability to mark and relocate cells/objects independently of stage position, so that registration between computer markers and the microscopic object is maintained when the objective lens is changed, when the stage is moved, or even when the slide is loaded on another HOME microscope. To determine the accuracy of the encoding stage system, several measurements were made with 10 x and 40 x objectives, using a small object on a conventional microscope slide, together with the England Finder slide
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Table 1 Error Components (in pm) of the Coardinate Measurement Using a Microscope Encoding Stage Equipped With Sony Transducers on the X and Y Axes and a England Finder Reference Slide x 10
x 40
X Y X Y Objective Sony encoder repeatability 1.0 1.0 1.0 1.0 2.9 1.3 2.9 1.3 Mean stage backlash 0.6 0.9 2.6 3.6 Max. display pixel error 0.6 2.2 0.6 Slide clip reload error (s.d) 2.2 Objective turret reload backlash 8.4 0 2.1 0 Sony encoder error 2.75 2.75 2.75 2.75 5 25 5 Max. England Finder error 25 6.7 20 Max, stage rotation setup error 6.7 20
for coordinate setup where appropriate. The results obtained in these tests are shown in Table 1. From this data, error distributions of object positions under various conditions can be computed as in Table 2. These computations suggest that the basic aim of unique registration of a typical cell nucleus (which is around 10 km in diameter) can be achieved on the prototype HOME systems when the location and subsequent relocation are carried out on the same microscope, with coordinate setup carried out with the same England Finder graticule slide (25.3 pm error with 40 x objective). However, larger errors ( k 3 7 km) may occur in cross-registration between different microscopes, due to differences in slide-clip rotation and variation in England Finder graticule slides. In practice, these errors are unlikely to prevent correct cell relocation in most practical situations because they are systematic, and the user will quickly adapt to their effect. However, user-controlled offset facilities and improved position encoding system are under development to increase the accuracy further.
Length Measurement Accuracy A test was carried out on a prototype HOME system to estimate the accuracy of the length measurement facility. The longest chord of a typical large muscle fibre was measured repeatedly (by a single operator) with different objective magnifications, and the standard errors of each set of measurements estimated. The results are shown in Table 3. At the centre of the field, the measurement means are very similar at all magnifications, but accuracy obviously increases as higherpower objectives are used. However, when the same object is measured at a far corner of the field, the measurements are substantially different. This error is due to differential distortion between the microscope and display images. Methods of software correction for this distortion are now being investigated. Potential Applications The basic HOME system is potentially useful in a wide range of applications [ll]. First, there are many applications for object marking facilities, either for ex-
Table 2 Overall Relocation Accuracy (in pm) Using a Microscope Encoding Stage Equipped With Sony Transducers on the X and Y Axes and a England Finder Reference Slide x 40
x 10
Obiective Without reload With reload (same microscope, no re-zero) With reload (same microscope, turret turned) With reload (same microscope, with re-zero) With reload
(another microscope)
X
Y
X
Y
4.0
4.0
2.1
1.9
4.6
4.0
3.8
2.0
9.6
4.0
4.4
2.0
8.5
5.5
5.3
2.6
37.3 30.0 37.1 29.7
Table 3 Length Measurement Accuracy (in pm) Using a Microscope Encoding Stage Equipped With Sony Transducers on the X and Y Axes Centre of the field Objective x 10 x 20 x 40
(n = 10) Mean Sd. 142.2 1.18 0.83 140.5 142.8 0.73
Top right corner (n = 10) Mean Sd. 131.9 132.6 134.0
2.36 0.78 0.49
amination by other personnel (e.g., regions of abnormality in tissue sections, cervical smear screening, and marking of cytogenetic abnormalities), or for recordkeeping purposes. Second, the system can be used to carry out morphometric measurements, either in general-purpose pathology use or for specific morphometric tissue-specific procedures (muscle, bone, and gut). Third, the system may be used for counting tasks (differential cell counts, mitotic index, and pycnotic index). Fourth, by displaying suitable grid patterns stereology applications can be implemented. Last, the system may be used for teaching purposes, either by displaying good examples of a particular cell type in a specimen, or for more advanced computer-aided instruction techniques. The addition of other facilities would allow the HOME system to be used for a n even wider range of applications. First, a motorized stage would permit much faster relocation of objects, which would be extremely valuable in some applications such a s cervical cytology screening. Second, the addition of a video camera would facilitate remote decision-sharing on difficult specimens or cells. Third, the provision of image analysis would greatly extend the range of measurements possible on manually selected cells or regions (in particular, DNA-ploi'dy and receptors) and would allow the introduction of fully automatic analysis of suitable objects in selected fields. Fourthly, networking facilities connecting several HOME workstations and data entry workstations, HOME automatic slide scanners equipped with motorized stage and image analysis facilities, and hospital information networks will greatly
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improve the information transfer in the routine pathology laboratory. Last, the addition of flexible self-proGarnming facilities would allow the pathologist to set up and test new measurement procedures and to export particularly successful procedures for general use.
ACKNOWLEDGMENTS The a u t h o r s w i s h t o express their indebtedness t o the p a thologist members of the HOME consortium: Pr. Etienne Martin (Bicstre, France); D r . A n n i e Morens (Grenoble, F r a n c e ) ; Pr. Peter Pfitzer (Diisseldorf, Germany); Pr.F r a n c o Rilke (Milano, Italy); Pr. Gerard Slavin (London, United Kingdom), and Pr. Peter Vooijs (Nijmegen, Netherlands); w i t h o u t their h e l p the project would n o t h a v e been possible. The a u t h o r s also w i s h t o express their grateful thanks t o individual m e m b e r s of the industrial p a r t n e r s in the project, in p a r t i c u l a r Dr. Nasse, Dr. Kohlhase, and Dr. Gausman of Carl Zeiss Gmbh, and D r . P. den Englese of Leitz Microscan, for their h e l p in designing the optical s y s t e m s for this project and also to Mr. P. Riley of Apollo Optical Services for his help in the design of the encoding stage and stage clip. The skillful assistance of Michele Brugal for documentation, Yolande B o u v a t for iconography, and Victoria von Hagen for m a n u s c r i p t preparation is acknowledged.
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analysis and expert systems integrated in medical microscopy. In: Proc. 9th Int. Conf. on Pattern Recognition, Roma (Italy). IEEE Computer Society Press, 1988, pp. 286-293. 3. Christiaanse JGM, Koper GJM, Verwoerd NP, Bonnet J , Ploem JS: A microscope for off-line relocation of LEYTAS detected objects. In: Abstracts of Analytical Cytology and Cytometry IX, Schloss Elmau, Germany, 1982, p. 42. 4. Dye R, Farrow ASJ, Fletcher DS, Tucker JH: Microscope Slide Clip. Patent Application No. 9019979.5, 1990. 5. Franklin WA, Bibbo M, Doria MI, Dytch HE, Toth J, DeSombre E, Wied GL: Quantitation of Estrogen receptor content and Ki-67 staining in breast carcinoma by the MicroTICAS image analysis system. Analyt Quant Cytol Histom 9:279-286, 1987. 6. Glaser EM, Van der Loos H: Computer Microscope Apparatus and method for superimposing an electronically produced image from the computer memory upon the image in the microscope field. US Patent 4.202.037. US Patent Ofice, Washington DC. 7. Gundersen HJG, Bendtsen TF, Korbo L, Marcussen N, Moller A, Nielsen K, Nyengaard JR, Pakkenberg B, Sorensen FB, Vesterby A, West MJ: Some new, simple and efficient stereological methods and their use in pathological research and diagnosis. APMIS 96: 379-394, 1988. 8. Schmitt P: Leitz MICROVID Modul zur Computer-Mikrospie. Ernst Leitz Wetzlar GMBH. 9. Seckinger D, Sugarbaker E, Frankfurt 0: DNA Content in Human Cancer. Arch Pathol Lab Med 1 1 3 5 1 9 4 2 6 , 1989. 10. Silverman JF, Finley JL, OBrien KF, Dabbs DJ, Park HK, Larkin EW: Diagnostic accuracy and role of immediate interpretation of fine needle aspiration biopsy specimens from various sites. A d a Cytol 33:791-796, 1989. 11. Von Hagen V, Morens A, Krief B: Highly optimized microscope environment: A new workstation for microscopic analysis in patholom. Anal Cell Pathol 3:249-254. 1991. 0”
