Journal of Neuroscience Methods, 1 (1979) 77--94 © Elsevier/North-Holland Biomedical Press

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COMPUTER-ASSISTED MAPPING WITH THE LIGHT MICROSCOPE

DONNA J. FORBES and ROGER W. PETRY

Department of Biomedical Anatomy and Department of Physiology, University of Minnesota-Duluth, School of Medicine, Duluth, Minn. (U.S.A.) (Received December 10th, 1978) (Accepted December 15th, 1978)

With the system described specific features in a histological section such as neuronal degeneration, specific cell types, or the silver grains of autoradiography can be accurately localized. It is simple to operate, and allows for data storage and retrieval. The system consists of a Zeiss Universal microscope equipped with stepping motor drives on the X and Y axes and interfaced to a PDP-12 computer equipped with a printer]plotter. The user controls the system through a joystick control box and teletype. In spite of magnification changes all points in the field remain constant relative to a point of origin selected at the beginning of the mapping sequence. Therefore, the outline and major features of a section are entered at low magnification while the detail of neuronal degeneration, for example, is added in its precise location at a higher magnification. The plotting of points can be done in either random fashion or in a systemized series of horizontal or vertical scans. A graphic display is available throughout the procedure and all maps are both printed on a Versatec printer/plotter and stored on a disk for future recall and analysis. Use of the system in plotting neuronal degeneration is described.

INTRODUCTION It is f r e q u e n t l y necessary t o m a p the precise l o c a t i o n o f m i c r o s c o p i c details such as n e u r o n a l d e g e n e r a t i o n , specific cell t y p e s or the silver grains o f a u t o r a d i o g r a p h y o n a m a c r o s c o p i c d r a w i n g o f a tissue section. Most o f the past m e t h o d s o f a c c o m p l i s h i n g this have been t i m e - c o n s u m i n g , i n a c c u r a t e t o v a r y i n g degrees, a n d limited in their application. T h e m o s t c o m m o n m e t h o d s have been t o m a k e a low m a g n i f i c a t i o n d r a w i n g with an o v e r h e a d p r o j e c t o r or t o m a k e a p h o t o g r a p h u p o n w h i c h the detail was later r e c o r d e d m a n u a l l y while viewing the tissue at high m a g n i f i c a t i o n . T h e s e m e t h o d s are especially t i m e - c o n s u m i n g and inaccurate. Use o f either the c a m e r a lucida or d r a w i n g t u b e is m o r e a c c u r a t e ; h o w e v e r , b o t h are i n c o n v e n i e n t since n e i t h e r allows for t h e m a g n i f i c a t i o n c h a n g e a n d t h e result is a large, c u m b e r s o m e drawing. In answer to these p r o b l e m s , a n u m b e r o f devices have been d e v e l o p e d w h i c h m e e t t h e i m m e d i a t e n e e d fairly well. T h e r e are n o w several versions o f the e l e c t r o n i c p a n t o g r a p h (Boivie et al., 1 9 6 8 ; Karten, 1 9 6 8 ; G r a n t and Boivie, 1 9 7 0 a n d others) w h i c h e m p l o y s p o t e n t i o m e t e r s a t t a c h e d t o the X and Y

78 axes of the microscope stage, which in turn drive the pens of an X--Y plotter. There also is the micromap (Patterson et al., 1976) which utilizes a laser coupled to the microscope stage and a series of mirrors to project the laser beam onto a drawing pad. Both the electronic pantograph and micromap allow for the magnification change necessary to switch from the outline drawing made at low magnification to a higher magnification where the microscopic detail can be distinguished and then mapped. However, there are limitations to both of these devices which have been overcome in the computer-assisted system described herein. In addition to these devices, it should be mentioned that a great many other non-computerized (McKenzie and Vogt, 1976) and computerized (Glaser and Van der Loos, 1965; Coleman et al., 1973; Reddy et al., 1973; Selverston, 1973; Wann et al., 1973; 1974; Boyle and Whitlock, 1974~ Llinas and Hillman, 1975; Ware and LoPresti, 1975; Capowski, 1977; Lindsay, 1977 and others) systems have been developed to analyze neuronal structures utilizing various techniques. Although these other computerized systems share many characteristics of the computer-assisted system described here, they are far more extensive and were designed to meet different needs. Utilizing a laboratory mini-computer coupled to a Zeiss Universal microscope equipped with stepping m o t o r drives on the X and Y axes, this system combines the capabilities of an electronic pantograph or micromap with the ability to store maps for later recall or analysis, erase errors during mapping and generate multiple copies of the printed output. Expansion of the system to include additional hardware and software is planned enabling analysis in many other applications. In this paper use of the system to map neuronal degeneration is described. A preliminary description has been presented before the annual meeting of the American Association of Anatomists (Forbes and Petry, 1978). CHARACTERISTICS OF THE MATERIAL TO BE EXAMINED Although it can be used for other purposes, this system has been developed in order to map neuronal degeneration. Since the characteristics of tissues stained for degeneration present certain difficulties during analysis which necessitate the mapping program, a brief description of this tissue follows. The tracing of neuronal connections by degeneration techniques is well known. Basically a neuronal pathway is lesioned so that the processes and terminals in question are separated from their parent cell bodies. The processes degenerate after an appropriate survival time and can be demonstrated in histological sections using one of the silver techniques of Nauta, Fink--Heimer or variations thereof. In the material used to develop the mapping program, a series of three 30 ttm sections of tissue were sampled at approximately 150 pm intervals.

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One section was stained with cresyl violet to demonstrate cytoarchitecture, a second section was stained with the Nauta or Fink--Heimer method and a third section was stained with the Nauta or Fink--Heimer m e t h o d followed by bleaching in K 3 F e ( C N ) J b o r a x and counterstaining with cresyl violet. This last series of counterstained sections was used for the actual mapping procedure in order to avoid any discrepancies or artifacts such as folds which might be encountered when switching between a cresyl violet and a silverstained section. For any section mapped, the companion cresyl violet section was used to confirm nuclear boundaries which might be obscured on the counterstained section. Likewise, whenever there was a question regarding the presence or extent of degeneration, the companion silver-stained section was consulted. When studying the material it is critically important to distinguish where the degenerating processes and their terminals are in relation to nuclear boundaries or cortical layers. Unfortunately, these cytoarchitectural features are often best observed at low magnification (Fig. 1) in a cell stain such as cresyl violet while the degenerating terminals can best be seen at higher magnifications (Figs. 2 and 3) with a Nauta or Fink--Heimer stain. Although degeneration is occasionally heavy enough to be seen at mid-range magnifications (Fig. 2), it is necessary to examine sections at high power (Fig. 3) to accurately locate the degeneration. Unfortunately, at the higher magnifications orientation relative to cytoarchitectural features is lost (Fig. 3). The present system deals with this problem by accomodating magnification changes at any point in the mapping sequence. Unlike the counting of autoradiographic silver grains which can be done automatically by computer (Boyle and Whitlock, 1974; Wann et al., 1974) the analysis of neuronal degeneration requires operator assistance throughout. The distinction between degenerating fibers and terminal degeneration, for example, is very subjective. In addition, there are other structures in the sections such as the reticular fibers in the walls of blood vessels which are also argyrophilic and, therefore, stain with the Nauta or Fink--Heimer methods. SYSTEM COMPONENTS

The mapping system utilizes equipment which is part of a semi-automatic image processing system assembled for the analysis of a variety of morphological problems. The components utilized in this application include an enhanced laboratory digital computer system, a computer-interfaced scanning stage light microscope, and the mapping system software.

Computer system Selection of CPU In theory, any mini-computer designed for laboratory research could func-

81

tion as the CPU (Central Processing Unit), since each has a fairly fast instruction execution time, hardware interrupt capability, and ability to be interfaced to a wide variety of peripherals such as graphic display screens, terminals, magnetic disk and tape storage, analog to digital converters, printer/ plotters, etc. This system utilizes a PDP-12 computer (Digital Equipment Corporation (DEC), Maynard Mass.) equipped with various components to be described below. The selection of computer for this application was immediate since the computer and interfaced microscope were already in house. It should be noted however, that the PDP-12, although specifically developed for the research laboratory, is no longer actively marketed by DEC due to high mainframe component and assembly costs. Fortunately, in recent years, scanning stage light microscopes have been interfaced to and the controlling software developed for more modern and available mini's such as the DEC PDP-11. Hardware The computer system, as illustrated in Fig. 4, contains 32K core memory, stepping motor interface, KWl2A programmable real-time clock, automatic priority interrupt (API), real-time point-plot graphics display, magnetic disk and tape storage, a Versatec D l l l 0 A printer/plotter (Versatec, San Diego, Calif.), teletype console, and several components not utilized in this application. The 32K memory contains the executing mapping program in 8K and buffer space for the map being plotted in 24K, accomodating a map comprised of up to 12,288 points. Under program control, the stepping motor interface generates electrical pulses which cause the stepping motors to move the stage one step per pulse. The KWl2A clock provides timing for the execution of specially enabled program instructions which trigger the stepping motor interface to 'pulse' the stage stepping motors, moving the scanning stage one step of 0.5 pm per pulse, up to a maximum of 200 steps/sec.

Fig. 1. Light micrograph of a frontal section through the left side of the squirrel m o n k e y thalamus. Various c y t o a r c h i t e c t u r a l l y distinct regions can be seen characterized by different cell types and arrangements; in s o m e cases the regions are demarcated by clear areas consisting o f unstained fibers. X indicates the location of one of three core samples taken from the tissue for a n o t h e r aspect of the degeneration study. Cresyl violet stain, x 14. Fig. 2. A mid-range magnification micrograph of squirrel m o n k e y thalamus showing degenerating fibers in a heavy band f r o m the lower left curving along the area indicated by the arrows. Additional scattered degeneration c a n n o t be seen at this magnification. X indicates the site of a core sample located in a position similar to that marked with an X in Fig. 1. Natau m e t h o d , x 56 Fig. 3. Higher p o w e r micrograph illustrating the character of degenerating nerve fibers in the squirrel m o n k e y thalamus. The arrow indicates a group o f typical degenerating fibers. Nauta m e t h o d , x 225

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The API allows for extremely fast processing of time critical events such as the stepping pulse timing and input/output (I/O) transfers. While the operator is tracing outlines and recording points, the graphics display screen displays the map as it is being created, providing the necessary spatial orientation for the operator. Program overlays are stored on disk for rapid read-in when activated. Partial and/or complete maps may be stored on disk and subsequently retrieved for modification, further development, or analysis. To conserve disk space, LINC tape storage is available for archiving. The printer/plotter provides copies of the maps as initiated by the operator through a command typed on the console teletype. The teletype console keyboard and joystick control box serve as the means through which all commands and stage movement directives are communicated to the mapping program.

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Microscope Mechanical The system utilizes a Zeiss Universal microscope (Carl Zeiss, Oberkochen, G.F.R.) equipped with a scanning stage with stepping motors attached to move the stage in the X--Y axes in 0.5 pm steps (Zeiss stage no. 47-34-81). The scanning stage has a maximum travel displacement of 75 mm × 25 mm in the X and Y directions, respectively. During setup, the operator manualiy positions the stage through the use of t h u m b wheels located under the stage. Once the mapping process has begun, movement of the stage is effected indirectly by manipulating the joystick whose indications are sampled by the program under clock control utilizing the analog to digital converter channels. As described in the previous section, the two stepping motors are actuated by the computer through the stepping m o t o r interface.

Optical The choice of microscope objectives is determined primarily by (a) the magnification at which cytoarchitectonic features are clear, and (b} the magnification at which the neuronal degeneration can be identified. An additional consideration is that the optical axis of each objective lens is usually not in precise alignment with every other lens. Therefore, at the present time a Zeiss 2.5X Pol Planapochromatic objective is used to outline the section and its major cytoarchitectonic features while a Zeiss 16X, 25X, or 40X Planapochromatic objective is used when mapping the neuronal degeneration. The 2.5X Pol lens can be aligned relative to any other single objective by a set of rings on the lens housing. Although the centerable feature is available on other higher magnification lenses, the 2.5X was chosen because of its lower cost and suitability to the present application. In practice, alignment is carried o u t by centering a small blood vessel beneath the crosshairs in the eyepiece using the higher power objective. Upon switching to the 2.5X objective, the centering rings are used to bring the blood vessel back to the centered position. The microscope also includes a variable magnification feature (Optivar) which allows for additional changes of magnification in multiples of 1.25, 1.6, and 2.0. One of the 12.5X eyepieces is equipped with a set of aligned crosshairs which are used as a reference point throughout the mapping program.

Software overview The principal mapping system routines are written in assembly language code (LAP-6) and assembled using the PAL12 assembler (C.G. R o b y , and D.J. Duffy, West Virginia University Medical Center) and executed through the OS/8 operating system. A semi-automatic image processing system (APAMOS} developed by Zeiss was acquired for the purpose of scanning

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Operation of the system as illustrated in Fig. 5 can be best explained in terms of: (A) initialization; (B) making the map; (C) map plot output; and (D) map storage, retrieval, and archiving.

(A ) Initialization Preliminaries The mapping session begins with the computer program greeting the user

85 and displaying instructions for general setup and lead connection verification.

Map specifications The user is asked if (a) a new map is being created, or if (b) one previously stored on disk is to be recalled for modification or further development. (a) New map. If the user indicates that a new map is to be created, the program instnlcts the user to determine a square size in millimeters which completely contains the area to be mapped, and to position the crosshairs at the lower right hand corner of the constructed square. Using the vernier scales located on the stage, the X and Y coordinates of this point are entered, establishing this position as the origin for the map. The operator then selects from a list of seven preprogrammed square sizes, a size large enough to contain the area to be mapped. (b) Recall a previously stored map. If the user wishes to recall a map already stored on disk, the filename is indicated, and the map is automatically loaded. The X and Y stage coordinates of the origin are displayed, and the program displays a reminder of the square size originally selected. With the correct slide in place on the stage, the operator manually locates the approximate position of the origin using the thumbwheels under the stage, and then fine adjusts to the exact position using the joystick. (B) Making the map Graphics display Upon completion of the preliminary and map specification steps, the role of the display screen changes from displaying instructions to displaying the map as it is created. (In the case of a previously stored map being recalled, the screen displays the map exactly as it was when filed.) The display is scaled to provide a full size picture of the area being mapped regardless of the selected square size. The origin is displayed as an illuminated point at the lower right corner. The location of the crosshairs, which also is displayed as a single point, is initially positioned at the origin. As the crosshairs are moved over the slide, so 'moves' the crosshairs dot on the display screen. Outlining in the trace mode The mapping session normally begins with 'tracing' the outline and section landmarks by directing movement of the crosshairs over these features. Through keyboard commands the operator indicates whether the computer is to simply 'move the crosshairs' over the slide as directed by the joystick w i t h o u t retaining its path, or if the traced path taken by the crosshairs is to be automatically recorded by the program. When in the trace mode and recording the path taken by the crosshairs, the outline trace is stored as a series of nearly adjacent points; the distance between the points is determined

86 by the c o m p u t e r as a function o f the size of the area to be mapped (Table 1) and the speed at which the operator traces. Thus, the course charted by the operator is stored as a series of poi nt coordinates and appears on the screen as a d o t t e d line marking the course of the crosshairs. A d o t t e d line rather than a solid line adequately serves to record the outline of the area of interest and also conserves the c om put er 's m e m o r y allocated for map storage. In the event the operator deviates inadvertantly from the intended course, the c o m p u t e r can be directed to ' r u b o u t ' the latest stored point(s) on the dot t ed line and automatically retrace its steps back to a point where tracing could again be resumed.

Plot mode Mapping the areas of degeneration on the outline tracing is accomplished through use o f the plot mode. While in the plot mode, point positions are recorded or 'marked' by depressing either B u t t o n M or the f o o t pedal. The plotting can be c o n d u c t e d in either (a) a random fashion or (b) a systemized scanning scheme.

(a) Random. When randomly plotted, areas of degeneration are typically identified by marking scattered degeneration with isolated dots, heavier degeneration as a series of closely spaced dots, and degenerating fibers as broken or u n b r o ken lines marking the course of the fibers. (b) Systemized scanning. To facilitate the localization of widespread degeneration and to avoid bias resulting from e x p e c t a n c y of location on the part of the operator, areas can be searched systematically in progressive scan sweeps (Fig. 6b). The sweeps, either horizontal or vertical in direction, may be spaced apart at selected distances as entered at the keyboard. T he minim u m distance between the adjacent scan sweeps is set to the resolution of the particular map. The process of mapping the degeneration through consecutive adjacent sweeps can be accomplished either manually, or with the aid of programcontrolled assistance from the computer. Under manual control, the operator determines the spacing and length of the sweeps on an individual basis, and may transfer freely between a systemized and random m e t h o d as desired. Program-controlled assistance permits l'apid consecutive sweeps over an area in a minimum a m o u n t of time. The operator initially determines the length, width, and location of an area to be searched and the spacing between the scan sweeps. The rectangular area to be scanned is, of course, completely contained within the boundaries of the originally selected mapping area and has an origin analogous to, but n o t necessarily the same as the origin o f the map. Once the area to be scanned has been defined, the c o m p u t e r positions the crosshairs at the origin of the area and awaits a p r o m p t from the operator to c o m m e n c e the progressive automatic scan. Once underway, the operator records the degeneration as the crosshairs pass

87

over it. If necessary, the scanning process may be interrupted and restarted through commands entered at the keyboard.

Miscellaneous The system offers the flexibility of freely transferring from one mapping mode to another at the convenience of the operator. Thus, a portion of the outline could be traced, some degeneration plotted, followed by more outline tracing, until the map is as complete as is desired. In addition to the 'rubout and retrace' feature described earlier, typing the 'alt mode' key on the teletype deletes recorded points {traced or plotted), one at a time, w i t h o u t causing the computer to retrace its steps. In this way any number of points may be deleted by repeatedly hitting the 'alt mode' key.

(C) Map plot output Hard-copy o u t p u t of the map can be initiated at any time with a keyboard command. Once initiated, the computer proceeds to transfer the map to the Versatec printer/plotter. As many copies as desired may be produced.

(D) Map storage, retrieval, and archiving A map, whether partial or complete, may be stored as a map file on disk for purposes of subsequent retrieval or archiving. All map files are stored .with unique operator assigned filenames and contain a header block and map storage block(s). When the computer performs the filing, it stores in the header block the X and Y stage coordinates of the map's origin and the map square size as were originally specified, and various housekeeping parameters necessary to the mapping program. Filed maps may be retrieved from disk for modification, further development or analysis {see map specification under Initialization). Archiving of maps onto LINC tape is a function p~rformed outside the scope of the mapping program. An OS/8 system program (FOTP) is routinely utilized to transfer files from one storage device to another. SYSTEM PERFORMANCE

The sample maps illustrated in Figs. 6a and 6b will be referred to in discussing the following performance aspects of the mapping system: the advantages and disadvantages of the random versus systemized scan plot modes, the time required to make a map, the resolution of the system, and the accuracy and repeatibility of the system. The maps in Figs. 6a and 6b were both made from a single frontal section of the thalamus of a squirrel m o n k e y in which a lesion had been made in the spinal cord 9 days prior to sacrifice. The 30 pm sections were stained

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Fig. 6. M a p s o f n e u r o n a l d e g e n e r a t i o n in a f r o n t a l s e c t i o n f r o m t h e r i g h t side o f t h e s q u i r r e l m o n k e y t h a l a m u s f o l l o w i n g a l e s i o n in t h e s p i n a l c o r d . T h e origin in t h e l o w e r r i g h t c o r n e r o f e a c h m a p is i n d i c a t e d b y t h e c r o s s h a i r s w i t h i n a circle. T h e X ' s i n d i c a t e t h e sites o f core s a m p l e s as s h o w n in Fig. 1~ t h e r e f o r e , n o d e g e n e r a t i o n a p p e a r s o n t h e m a p s at t h e s e sites, a: p l o t t i n g o f p o i n t s d o n e in t h e r a n d o m p l o t t i n g m o d e ; b: p l o t t i n g o f p o i n t s d o n e in a series o f h o r i z o n t a l s y s t e m i z e d s c a n s .

89 with the Fink--Heimer m e t h o d and counterstained with cresyl violet. Since the tissue section was approximately 8 X 10.5 mm, a 16 m m plotted square size was selected. The outline for each map of the section is the same; it was stored on disk before entering the plot mode and was then recalled to plot the points shown in Figs. 6a and 6b. The map in Fig. 6a was made using the random plotting mode while the map in Fig. 6b was made using the horizontal scanning mode with a distance of 5 increments or 80 pm between adjacent scan lines. In comparing the two maps it should be noted that there is generally very good agreement regarding the location of areas of degeneration and that it is possible to describe the location of the degeneration relative to various cytoarchitectural boundaries. There are variations in the plotted points shown in Figs. 6a and 6b, which reflect various aspects of the random and systemized scanning plot modes. The advantages and disadvantages of both types of plot must be weighed before deciding on their respective use. The random plot produces a map which looks more like the actual pattern of degeneration since the operator can follow the course of the degenerating fibers, and is able to mark fragments of degeneration which might otherwise be located between the adjacent scan lines of a scanning plot. However, there is a tendency on the part of the operator using the random plot to cover only those areas where degeneration is expected. Thus, small fragments of degeneration in adjoining areas may be totally missed. This can be seen in the sample maps since there are areas in Fig. 6a which appear to be totally free of degeneration but which are shown in Fig. 6b to actually contain some degenerating fragments. As will be discussed in a subsequent section, this operator bias does make the random plot faster than the scanning plot since a smaller overall area is generally covered. The advantages of the systemized scan are two-fold. First of all, the bias of operator expectancy is moderated to a great extent. Once the outline is traced, the operator no longer needs to be concerned about location of the crosshairs on the section and can concentrate on characterizing the degeneration under the crosshairs at any given moment. As a result more of the section is usually scanned and more degeneration is usually found than when making a random plot. The second major advantage of the scanning plot is that back-tracing is eliminated since the operator always knows the area t h a t has been already covered. This saves time and prevents double marking of the same fragment of degeneration. As described in the next section, the major disadvantage of the scanning plot is that it takes longer than a random plot. The minor disadvantages that the plot looks less realistic and that the operator cannot follow a degenerating profile out of the path of the scan can be alleviated somewhat by a combination of the scanning and random plots. Since it is very simple to switch back and forth between the random and scanning plot modes, the operator can actually use the scanning plot to keep approximate track of where plotting has been done, but periodically change to the random plot to add detail which would otherwise fall between adjacent scan lines. Decreasing the distance between scan lines in those areas

90 where degeneration is heavy would also produce a more realistic-looking map. Although bot h horizontal and vertical scanning are possible, horizontal generally has been used with the degeneration material. In those situations where the tissue section might be oriented differently on the slide, the vertical option could be utilized. The time required to make a map depends primarily on the chosen square size and the a m o u n t of detail recorded in both the trace and plot modes. Since the stepping m ot or s can move no faster than 0.1 mm/sec, the larger the selected square size, the longer it takes to traverse the distance selected. For example, with a 32 m m square size it takes slightly more than 5 rain to travel across a side of the square, whereas with a 0.5 m m square it takes only 5 sec. The a m o u n t o f detail recorded and, therefore, the time required can vary tremendously. In the trace mode one can easily outline a section in 10 min, b u t if every conceivable morphological detail were added the time required would be e x t e n d e d ad infinitum. It t o o k approxi m at el y 15 min to make the outline tracings in Figs. 6a and 6b. In the plot mode, time becomes a factor in choosing between a r a ndom and a scanning plot. In a random plot a section such as that shown in Fig. 6a with a 16 mm square size requires about 45 min to plot the points after making the outline trace. In a scanning plot the choice of distance between adjacent scan lines directly affects the time required for the plot. For instance, not e the example of Fig. 6b, in which a piece of tissue approximately 8 X 10.5 mm was plotted using a 16 mm square size. Although n o t true for this particular plot, if each scan were to have traversed the entire 8 m m width, 80 sec would have been required for each scan. With 10 increments or 160 pm between adjacent scan lines, a total of 1.5 h would have been required to cover the entire 10.5 m m height of the section; at a spacing of 5 increments or 80 pm, as illustrated, approximately 3 h would have been required. In reality, the entire width and height of a section are rarely scanned so that the plot in Fig. 6b actually t o o k about 2 h to complete with the outline drawn previously. Time becomes a factor in the choice between random and scanning plot modes since the latter in almost all cases takes longer. However, since the scanning plot succeeds in covering m or e of the tissue section and actually marking mo r e points of degeneration, it is probably more accurate than the r a nd o m plot. Thus, the final decision between random and scanning plot m od e must include consideration of the relative importance of the time required for the plot versus the accuracy desired. Table 1 lists the selection of mapping area sizes and the associated plot resolutions. The resolution of the generated map is a function of the resolution and scan line size of the printer/plotter, the resolution of the stepping motors, and the size of the area to be studied. As m e n t i o n e d earlier, the Versatec D l l l 0 A p r i n t e r / p l o t t e r produces plots via a raster scan transfer similar to that of a television picture. The horizontal scan line of the D 1110A contains 1024 point positions. The vertical resolu-

91 TABLE 1 C O R R E L A T I O N O F R E S O L U T I O N WITH S I Z E O F A R E A S T U D I E D P l o t t e d s q u a r e size a ( m m ) R e s o l u t i o n in t r a c e m o d e (variable) (tim) R e s o l u t i o n in p o i n t / p l o t m o d e (fixed) (/lm)

0.5 1 0.5

1 2 1

2 4 2

4 8 4

8 16 8

16 32 16

32 64 32

a P l o t t e d square size is t h e d i s t a n c e along o n e side o f t h e square o f tissue studied.

tion is equivalent to the horizontal (100 dots/in.) enabling the printer/ plotter to produce a spatially accurate plot square or plot matrix of 1024 X 1024 dot positions. The map becomes a transformation of mapped or 'plotted' points onto the 1024 X 1024 matrix. The length of a side of the selected square mapping area is divided by 1024 to obtain the 'map resolution' or the smallest distance between definable features on the slide. This measure of distance is called the 'stage increment'. In order to fit a map onto a single 1024 X 1024 dot matrix there can be at most 1024 stage increments of travel from the origin in either the X or Y axes. Given that the pulsed step of the stepping m o t o r is 0.5 pm, the smallest distance traveled by the stage as directed by the computer for one stage increment will be a certain multiple of 0.5 pm as determined by the size of the mapping area selected by the operator. Since 1024 stage increments of 0.5 pm sum up to only a little more than 0.5 mm, larger mapping areas are obtained by taking multiples of the 0.5 pm step to form the stage increment. Thus, resolution of the generated map must be sacrificed to gain the larger mapping area. To minimize computational time during execution of the time-critical portions of the computer program, only selected multiples of the 0.5 pm step are utilized. More precisely, stage increments consisting of exactly 1, 2, 4, 8, 16, 32, or 64 pulsed steps have been established, providing corresponding map resolutions of 0.5, 1, 2, 4, 8, 16, and 32 pm, respectively, in the plot mode. Recalling that a dotted line is utilized in the trace mode to conserve memory, resolution in the trace mode can be no better than half the possible plot mode resolution. The accuracy and repeatibility of maps made with this system depends upon the proper preparation of the tissue, the operator's judgment and which plot mode is utilized. The tissue preparation and operator ability apply to degeneration material whether analyzed by this system or any other. Consistent and careful preparation of the tissue avoiding bad staining, cracks, creases and folds is very important. Likewise, the operator must be able to judge with reliability the boundaries of cell groups or other morphological landmarks when making the outline of a section and the presence or absence of degeneration in a given area when plotting points. Human error is inevitable and must be assumed to some extent when considering the accuracy of a map. As mentioned earlier, the accuracy of the scanning plot mode is probably greater than that of the random plot mode. This is due to

92 the fact that in the former, operator expectancy is minimized, generally a larger area is covered, more scattered areas of degeneration are found, there is no backtracking and double marking, and the operator can devote full attention to characterizing the degeneration without being concerned with the actual location of the crosshairs on the section. DISCUSSION Using the system herein described, microscopic particles such as neuronal degeneration can be localized with considerable accuracy relative to identifiable morphological features of a tissue section. In comparison to other systems available for .this type of analysis, the computer-assisted mapping system has several advantages. Although the electronic pantograph and micromap are certainly great improvements over previously available methods in terms of accuracy, savings of time, and the handling of magnification change, they do have certain limitations which the computer-assisted system overcomes. For example, at any time during the course of a plot, points can be removed if a tracing or plotting error occurs; with the pantograph or micromap these errors remain. In addition, with the computer-assisted system it is possible to produce any number of hard copy printouts either during or after a plot. Thus, one could create a progressive series of maps in which different cell types or the degeneration in different nuclei are added in sequential fashion. The multiple printouts at the end are useful in providing both working copies and good quality final copies which can be directly labeled and photographed for illustration purposes. One of the biggest advantages of the computer-assisted system versus the pantograph or micromap is that this system provides for data storage and retrieval. If the operator is interrupted while mapping, for instance, the map can be stored for later recall and completion. This flexibility adds to the convenience of the system since the operator does n o t have to be assured of a fixed block of time in which to complete a map. It is also possible to store the traced outline of a section separately from the complete map. This was done in generating Figs. 6a and 6b in order that the outlines of the section would correspond precisely to one another. It would also be possible to plot different cell types or the degeneration in separate nuclei on separate but identical outlines of a section. Another advantage of the computer-assisted system is that it allows for expansion to other applications. As the system presently stands, it can be used in any situation where there is need to localize some very small feature on a histological slide relative to some larger boundary or landmark of the section. However, for many of the proposed applications it would be helpful if the present system could be expanded and improved. For example, in an autoradiography study using tritiated thymidine, presently underway in the laboratory, it would be useful to record for each labeled cell not only its location as can be done at present, but also such ancillary information as cell

93 size, grain c o u n t or cell type including separate printed symbols for each cell type. In another study there is need to localize a cytoarchitectural area of interest under low power and then to characterize the cell types in that area under high power. A plotted square size of 32 mm is required to encompass the entire section and thus the resolution is only 32 pm (Table 1). In order to characterize the cells the resolution must be improved to perhaps 0.5 or 1 pm. Thus, a small 0.5 or 1 m m square with a new origin related by the computer to the origin of the original section could be chosen which would encompass the area of interest but be enclosed within the original square. Thus, both the needed resolution and the accurate localization would be achieved. Other improvements considered for the system include computer-adjusted centering of the objective lenses and use of other inputs such as a data tablet. Many of the proposed improvements or added capabilities are already part of the sophisticated computer systems mentioned in the introduction which are used to anaIyze neuronal structure. Although the present system was primarily designed to replace and improve upon the electronic pantograph, it does utilize m a n y of the basic features of these other computerized systems. Thus, anyone having access to one of the computerized systems might consider utilizing the features which have been described here to assist in mapping with the light microscope. ACKNOWLEDGEMENTS The authors are grateful to Dr. Robert S. Pozos for making the computer facilities available, to Linda Smedberg for technical assistance, to Susan Radzek for illustrations, and to Sandy Nyberg for typing the manuscript. Funds for the program development were provided in part by a University of Minnesota Graduate School Grant to Dr. Forbes. REFERENCES Boivie, J., Grant, G. and Ulfendahl, H. (1968) The X--Y recorder used for mapping under the microscope, Acta physiol, scand., 74: 1A--2A. Boyle, P.J.R. and Whitlock, D.G. (1974) The application of a computer controlled microscope to autoradiographs of nerve tissue, Abstr. Decus. Proc., 95--99. Capowski, J.J. (1977) Computer-aided reconstruction of neuron trees from several serial sections, Comput. biomed. Res., 10: 617---629. Coleman, P.D., West, M.J. and Wyss, U.R. (1973) Computer-aided quantitative neuroanatomy. In B. Weiss (Ed.), Digital Computers in the Behavioral Laboratory, Appleton-Century-Crofts, New York, pp. 370--426. Forbes, D.J. and Petry, R.W. (1978) A semi-automatic computer system for mapping under the light microscope, Anat. Rec., 190: 597. Glaser, E.M. and Van der Loos, H. (1965) A semi-automatic computer-microscope for the analysis of neuronal morphology. IEEE Trans. biomed. Engng, BME-12: 22--31. Grant, G. and Boivie, J. (1970) The charting of degenerative changes in nervous tissue with the aid of an electronic pantograph device, Brain Res., 21 : 439--442.

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