AMIEZ, G. (1987) Sur deux probl6mes concrets en calcul scientifique: homog6n6isation des plaques nervur6es et cicatrisation des ulceres. Thesis, Besan~on, No 41, 26th June. A~IEz, G. (1988) Cicatrisation des ulc6res. Congr6s National d'analyse num6rique, Port Barcar6s, 12-14. CARRELL, A. and HARTMANN,A. (1916) Cicatrisation of wounds. The relation between the size of a wound and its rate of cicatrisation. J. Exp. Med., 24, 429-450. ERIKSSON, G., EKLUND, A. E., TORLEGARD,K. and DAUPmN, E. (1979) Evaluation of leg ulcer treatment with stereophotogrammetry. Br. J. Dermatol., 101, (2), 123-131. HASmMOTO, K. (1974) A new method for surface ultrastructure, comparative studies of scanning electron microscopy, transmis-

1 Introduction MEASUREMENTS OV electrical potentials inside living cells is accomplished using microelectrodes made from glass capillary tubing which has been heated and pulled to obtain a capillary with a diameter of 1/~m or less. The capillary is filled with sodium chloride solution, inserted into a holder and connected via a silver chloride contact to a very high impedance amplifier. Using a precision three-dimensional manipulator the microelectrode is positioned such that it penetrates a cell of a tissue specimen immersed in a nutrient solution in a tissue perfusion system. The degree of difficulty associated with obtaining a microelectrode measurement is inversely proportional to the size of the cell upon which the measurement is being carried out (KuRIYAMA and ITo, 1975). The development of a three-dimensional microprocessor-controlled electrode-positioning system for microelectrode measurement has been motivated by the need to minimise the time required to position the microelectrode and to obtain successful microelectrode measureCorrespondence should be addressed to Dr Nelson G. Durdle at address 1. First received 10th September 1990 and in final form 29th May11991 9 IFMBE: 1992

Medical & Biological Engineering & Computing

sion electron microscopy and replica method. Int. J. Dermatol., 13, 357-381. MAKK], S., AGACHE, P., MIGNOT, J. and ZAHOUANI,H. (1984) Statistical analysis and three dimensional representation of the human skin surface. J. Soc. Cosmet. Chem., 35, 311-325. MIGNOT, J., CHUARD, M. and ZAHOUANI, H. 1985 Microtopographical analysis of human skin surface. Bioeng. Skin., 1, 101-110. PERDRUPT, A. (1987) The healing rate in leg ulcers. Acta Derm. Vener., 52, 136-140. ZAHOUANI, H. (1989) Quantification de la topographie des surfaces. Application h la peau humaine et h ses pathologies. Thesis, Besanqon No 115.

ments from smooth muscle in vitro tissue specimens from the gastrointestinal tract. The cells of this smooth muscle are spindle shaped with a diameter of 2 - 4 #m and a length of 0.2-0-4mm. Because the cells are very small and because they are continually contracting it is very difficult to successfully penetrate them and obtain microelectrode measurements before the electrode is broken by cell contractions. To facilitate measurements in these cells a partially automated system is required. Because of the 2-4 #m cell diameter such a system would have to permit positioning of the electrode to within 0.5/~m and position the electrode free of vibration. Electrode vibratio~ would either cause a loss of cell penetration or breakage of the electrode. No such automated system is available commercially. This technical note describes a microprocessorbased system designed to make the required microelectrode measurements. The position of the microelectrode in XYZ space is determined by three stepper motors which are controlled via a three-dimensional joystick. If the number of steps to be taken by the stepper m o t o r is large, the stepper motor control algorithm will accelerate/decelerate the motors at their m a x i m u m rate until the electrode comes to within a few micrometres of the desired position, then rotate with constant velocity until the final position is reached. This

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arrangement minimises the time required and prevents the motors from either slipping or overshooting (ATHANI and DESHMU~:H, 1979). The system monitors the microelectrode signal to detect penetration when the potential drops to the intracellular potential (typically - 8 0 to -90mV). When cell penetration is detected the system disables XY movement to prevent accidental breakage of the electrode. Additional features of this system include the control of the microelectrode via a keyboard, automatic 'home' position control, automatic halting of the microelectrode movement when the expected intracellular potential is obtained and great flexibility in altering the control parameters. Emphasis has been placed on reliability and ease of operation. 2 Description of t h e system

2.1 System requirements The three-dimensional electrode-positioning system controls the position of the microelectrode in steps of 0-5/~m and reliably displays the microelectrode position in the XYZ space with ease and efficiency, with an accuracy of 0.5 ~tm with as much flexibility as possible. Because of the many difficulties associated with smooth muscle intracellular measurements, the apparatus must be easy to operate. The system design must be such that vibration or mechanical shock has no effect on the position of the microelectrode. It is desirable that the system be easily reconfigured to maximise its possible applications. A block diagram of the developed system is shown in Fig. 1.

2.2 Implementation The complete three-dimensional microprocessorcontrolled electrode-positioning system can be described as comprising five components: (a) (b) (c) (d)

the microcomputer the stepper motors' control circuitry the keyboard input interface and display circuitry the analogue processing module (three-dimensional joystick interface) (e) the output module, comprising hydraulic interface and precision three-dimensional manipulator with electrode holder. The complete system was constructed on two circuit boards: a digital board containing the microprocessor with its associated circuitry and the analogue board containing the stepper motor interface, joystick interface and signal amplification and processing circuits. 2.3 Microprocessor system A block diagram of the dedicated microcomputer system is shown in Fig. 2. The system uses a Motorola MC6809 microprocessor utilising a 1 MHz clock. Two Motorola asynchronous communication adaptors MC6850 (MELEAR, 1981) are used to create serial RS232-C ports for connection to a terminal and a host computer system. The Motorola 6809 monitor program facilitated communication between the terminal and the host. All software was developed using the Motorola 6809 cross-assembler

Fig. 1 Blockdiagram of the microprocessor-controlled electrode-positioning system 240

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clock ~I~ and power-up reset

keyboard entry to operate in an asynchronous data entry mode (National Semiconductor, 1977). The main input functions are listed below.

MC6809

microprocessor

(i) (ii) (iii) (iv) (v) (vi) (vii) (viii) (ix)

(4k)

contro[ bus data bus address

J

busj

~t_ EPROM -- ~

(4k)

"3

--~

PIA2

interface to keypad, display stepper motors analogue data aquisition system beepers and buzzers

R5232-C link ACIA (T) ~ RS232-C

ACIA (H) ~-

Zl -I

link v

set minimum intracellular potential threshold set maximum intracellular potential threshold set microelectrode x-position set microelectrode y-position set microelectrode z-position automatic 'home' position 'enter' a 'clear' function reset 'go' function (enable system to start scanning joystick input) (x) digits 0-9.

Keyboard entry enables the user to quickly position the microelectrode to the desired position. This mode disables the joystick control mode, which is used to control the final precise positioning of the microelectrode. The display consists of four rows of four digits of sevensegment LED displays with a fixed decimal point. The display is controlled using two Intersil ICM7218A display drivers (Intersil, 1986). The parameters normally displayed are as follows: (a) (b) (c) (d)

PTM

microelectrode position in the x-direction microelectrode position in the y-direction microelectrode position in the z-direction intracellular potential.

Fig. 2 Blockdiaoram of the microcomputer running on an Amdahl 470 system. The two ports remain on the system to permit future upgrading of the software. The system program resides in a 4byte E P R O M and the system RAM comprises two 2byte x 8 C M O S 6 1 1 6 RAMs. The system also uses two Motorola MC6821 parallel interface adaptors (PIAs) and a Motorola MC6840 programmable timer (CARR, 1982). 2.4 Stepper motor control circuitry The stepper motor control circuitry consists of parallel interface adaptors and stepper motor transistor drivers. Stepper motors controlling movement in the x- and ydirections are controlled by the output lines on the port B side of a parallel interface adaptor (PIA 1) and the stepper motor controlling movement in the Y-direction is controlled by the four output lines on the port B side of a second parallel interface adaptor (PIA 2). These lines are pulsed in sequence to cause the motors to rotate in either clockwise or counterclockwise direction. A motor's speed is controlled by the rate at which the motors are pulsed. The stepper motor control algorithm is such that if the number of steps to be taken by the motor are large (large displacement of the microelectrode), the motors will accelerate/decelerate until a position within a few micrometres of the desired microelectrode position is reached and then rotate at a constant velocity to the final position. This arrangement minimises the time required to position the microelectrode. 2.5 Keyboard and display circuitry The keypad is used in an interrupt mode and inputs control parameters for the control of the three stepper motors through a 4 x 4 matrix keypad. The keypad is interfaced to the microcomputer system via a CMOS 16-key encoder. This interface arrangement enables the Medical & Biological Engineering & Computing

2.6 Analogue processing module The analogue processing module acquires data for the control of the three-dimensional manipulator. The joystick is used for continuous control of the microelectrode position in xyz space. The joystick is connected to the microcomputer system via a 4-to-1 analogue multiplexer, sample-and-hold circuitry and an eight-channel analogueto-digital convertor. Another function of this module is the monitoring of the intracellular potential via channel 4 of the analogue multiplexer. This signal is compared with the preset threshold level and is used to stop the advancement of the microelectrode into the cell when the required signal level is obtained. 2.7 Output module The output module shown in Fig. 3 consists of (i) a stepper motor-to-hydraulic system interface (ii) hydraulic link (iii) three-dimensional hydraulic-controlled manipulators with microelectrode holder. The three stepper motors are linked to the hydraulic system via three high-precision micrometers. This arrangement enables the circular motion of the stepper motors to be converted to linear motion for the control of the hydraulic system. The hydraulic system comprises 6.4mm (0.25in) diameter master bellows mechanically linked to the micrometers and 25-4mm (1-0in) diameter slave bellows attached to the three-dimensional manipulators with flexible tubing linking the master and slave bellows. This novel arrangement first used in our laboratory isolates the manipulators with the microelectrode holder from the controller and stepper motors apparatus, thus overcoming any vibrational problems. With this arrangement the microelectrode can be positioned in steps as small as 0.5~m.

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Fig. 3

Diagram of the stepper motor hydraulic control system

3 Performance The system was tested both in joystick and keyboard mode. Because absolute positioning is not one of the system requirements, only the accuracy and reproducibility of relative displacements needed to be tested. For this purpose a high magnification ( • 200) microscope with calibrated scale was used to measure the microelectrode movement in the x-, y- and z-directions. The range of control is from 0 to 128/tm with a resolution of 0.5/~m in all three directions. Hysteresis was too small to be observed and measured using the test microscope. The

make and maintain smooth muscle microelectrode measurements. No such degradation in recording performance occurred with the system installed. A typical recording obtained from two different types of colon smooth muscle cells is shown in Fig. 5. 4 Discussion

o~

4.1 Hardware considerations The Motorola MC6809 microprocessor was chosen primarily because of the software development support available for this processor. Significant hardware support for the processor was provided by the keypad encoder, display driver and the stepper motor interface. This minimises the supervisory tasks of the processor and provides sufficient time for execution of the control algorithms.

_~

4.2 Software considerations The software comprises two modules:

125

s o

100

2s

0

50

]50

lO0

200

250

relQtive x - stepper motor input, steps

Fig. 4

Plot of relative electrode position in the x-direction against x-stepper motor input units

position of the electrode in all three directions was reproducible when the electrode was returned to the 'home' position and then moved to the limit of its range. A plot of electrode movement against position input (in stepper motor steps) is shown in Fig. 4. Mechanical shocks due to discrete steps of the stepper motor were not observable under a high-power microscope. Also, such shocks or any vibration added by the system would have had a negative impact on the ability to

~-- 2os IIl|lllllllllllllll|lllllllllllllllllllllllllllllilllllll

-100mV - -

F- 3os -.1 IJlJllllllJlJJllllllJll]llilllllllllll

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-100mY--

.~ b

Fig. 5 Intracellular potential measurements recorded from (a) the circular smooth muscle layer and (b) the longitudinal smooth muscle layer of the canine colon 242

The main control program occupies approximately 2k bytes of E P R O M and facilitates the control of the three stepper motors via the joystick. The interrupt service routine also occupies approximately 2 k bytes of E P R O M and enables control of the three stepper motors via the keyboard. The programs are modular and designed to permit easy modification to accommodate system hardware changes. The stepper motors' control algorithm is such that it allows two modes of operation, namely: (i) constant speed operation (ii) acceleration/deceleration speed operation. The motors' stepping algorithm is as follows:

"1

OV--

IIIHIll

(a) the main control program (b) an interrupt service routine.

(a) The motor is started at a stepping rate somewhat below the start-stop rate so that the motor starts off smoothly without missing any steps. (b) The motor is then accelerated to its slewing rate (maximum rate without slipping). (c) The motor executes the major portion of N steps at the slewing or maximum rate. (d) When the required end position approaches, the motor is decelerated down to the start-stop rate. (e) The motor operation is stabilised at the start-stop rate before stop so that the motor halts after exactly N steps without overshooting. In the joystick mode the motor control algorithm is such that the microelectrode follows the path of the three-

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dimensional joystick. The automatic 'home' routine enables the microelectrode to return to its original position by first moving in the z-direction followed by the x- and y-directions. This scheme prevents breaking of the microelectrode by ensuring that the microelectrode is out of the tissue before it moves in the x y plane. When the microelectrode is inside the tissue both the x and y direction movement is disabled. This ensures no damage to the microelectrode or the cell under test if the joystick setting is accidentally altered.

5 Conclusion A three-dimensional electrode-positioning system has been constructed for microelectrode measurement and the various components of the hardware have been successfully tested either directly or indirectly. The mechanical system, comprising stepper motor interface to the hydraulic system, hydraulic link, three-dimensional joystick and the three-dimensional manipulator with microelectrode holder, gave the required three-dimensional submicrometre control accuracies. In checking the system operation it was determined that

1 Introduction

STRAIN GAUGES are accurate, durable, linear, relatively inexpensive, have long-term repeatability and little or no hysteresis. In addition, they are inherently simple transducers to use. However, despite their many advantages, strain gauges typically require milliwatts of power for their operation. This power requirement makes strain gauges inefficient for practical use in many battery-powered prosthetic limbs where 'micropower operation' is desirable for standby electronic instrumentation. We define micropower as power less than 1 mW. This technical note describes a micropower strain-gauge sampling circuit (MSGSC) that has been developed to interface with a strain-gauge bridge and that reduces operating current by supplying the bridge with pulsed power. The resulting operating current of the strain-gauge bridge Received 16th May 1991

9 IFMBE:

1992

Medical & Biological Engineering & Computing

the Motorola MC6809 microprocessor is heavily used at its 1 MHz clock frequency. Thus the MC6809 was a good choice in terms of processing power. The development of this instrument makes a significant contribution to the gastrointestinal research for which it was designed. Also, the design has sufficient versatility that it could be a significant asset in any physiological or biological experiment requiring microelectrode positioning in submicrometre increments. References ATHANI, V. V. and DESHMUKH,R. M. (1979) Microprocessor control of stepper motors. Proc. IEEE Int. Conf. on Micro & Minicomputers, Houston, 14th-16th Nov. CARR,J. J. (1982) Desiynin9 microprocessor-based instrumentation. Reston, New York. lntersil (1986) Intersil integrated circuit data book. KURIYAMA,H. and ho, Y. (1975) Recording of intracellular electrical activity with microelectrodes. In Methods in pharmacoloyy, Vol. 3 Smooth Muscle. DANIEL,E. E. and PATON,D. M. (Eds.), Plenum Press, New York. MELEAR,C. (1981) An intelligent terminal with data link capability. Motorola Application Notes, AN830-1981. National Semiconductor (1977) MSS/LSI data book. National Semiconductor.

is proportional to the duty cycle of the pulsed power supply. The combined operating current of the MSGSC and strain-gauge bridge, that we have developed, is less than 100/~A over a supply voltage range of 3-0-12.0V. Some pulsed-power strain-gauge circuits have been previously described, but the gauges were usually pulsed with hundreds of volts in an effort to increase their strain sensitivity while maintaining acceptable power dissipation (ARLOWE, 1974; DRAPER, 1984; ILTIS, 1970; POLESHCHUK, 1982; SKOTNIKOVand SER'EZNOV,1970; STEIN, 1958; 1965). The circuit was designed to allow strain gauges to be used in prosthetic limbs without excessive battery drain. In particular, this circuitry was developed to allow strain gauges to be used for force transduction in an extended physiological proprioception (EPP) control system as described by DOUBLER and CHILDRESS (1984a; b). Specific details concerning the interfacing of the MSGSC with the E P P controller will be discussed later in this note. The general control concept of E P P was derived from the original work of SIMPSON(1974).

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