0 Journal of Microscopy, Vol. 103, Pt 2, March 1975, pp. 265-270. Received 17 September 1974; revision received 13 November 1974
Short Technical Note A robust micromanipulator for the scanning electron microscope
by J. B. PAWLEY* and A. BOYDE,Department of Anatomy and Embryology, University College, Gower Street, London WC1E 6BT SUMMARY
A simple device for holding and moving mechanical tools in the region of a sample being viewed in the scanning electron microscope is described. The unit has a 20: 1 mechanical reduction and when fitted with a tungsten carbide dental chisel, it is sufficiently rigid to cut biological hard tissues. Alternately, when fitted with an electro-etched tungsten needle, it can be used, in conjunction with specimen stage controls, to remove individual cells from the surface of soft tissues. Examples of these applications are illustrated. INTRODUCTION
Several devices have been developed to make possible the controlled mechanical disruption being viewed in the scanning electron microscope (SEM) (MacAlear, Germinario & Fucci, 1971; Pawley, 1972; Pawley & Nowell, 1973). These have been of two general kinds. In the first, relative motion between tool and the sample surface is provided by the use of the controls normally used for positioning the sample, while in the second such relative motion is provided by movement of the tool itself. Both approaches have their advantages. The first, lacking the need for fine mechanical motions, is easier to construct and also has the added advantage that the area of greatest interest where the tool touches the sample, remains in a fixed position on the SEM display screen. On the other hand, the second approach offers a more familiar feel for intricate operations because we normally move the tool and not the workpiece in everyday life. Also, motions in directions not parallel to the axes of stage motion are more easy to perform. This paper describes a micromanipulator which embodies some aspects of both approaches. It was primarily developed to hold various tools so that the process of cutting dental tissues could be viewed in the SEM. As these materials are very hard, the rigidity of the tool-holding end of the manipulator was a major concern. Of secondary importance was the desirability of being able to make co-ordinated motions in at least two dimensions with sufficient precision to be useful at low magnifications ( x 50-400) while leaving fine relative motions to be performed by the stage motion controls.
* Present address : Department of Veterinary Anatomy, University of California, Davis, California, U.S.A. 265
J . B. Pawley and A . Boyde DESIGN
The result is shown in Fig. 1. The design utilizes as a tool support a 9.5 mm diameter stainless steel shaft (s) whose axis intersects the electron optical axis at a working distance of 8 mm. This pivots on a ball-joint made from a 26 mm ballbearing @) which is sealed to the shaft and to the outer tube (ot) by O-rings (or) and held in place by atmospheric pressure. The outer tube is soldered to a standard Stereoscan side plate (sp). Motion of the tool in the directions perpendicular to the axis of the shaft is produced by a lockable two-dimensional mechanical manipulator (yz) and utilizing a 10: 1 pivoting-level reduction* and ball bearing slides (y and z) and attached to the outer tube with a metal plate. Because of the relative sizes of the level arms on either side of the pivot point the overall mechanical reduction was 20:l in directions perpendicular to the shaft axis. The manipulator is connected to the inner tube (it) which surrounds the shaft by a special collar (rc) which uses four straight pieces of spring wire (w) to provide a small mount of flexibility in order to prevent binding as the angle between the shaft and the manipulator varies during shaft movement. The collar can be rotated in the clamp where it is attached to the manipulator permitting the tool angle to be varied and then fixed by using a locking screw (rl). Movement of the tool in the third orthogonal direction, that parallel to the shaft axis, is provided by a screw (X) which moves the shaft with respect to the inner tube. Relative rotation between these last two components is prevented by a pin sliding in a slot (ss). Atmospheric pressure on the end of the shaft provides ‘spring’ loading to remove back-lash in the screw drive. The tool (t), e.g. a dental bur or an electro-etched tungsten needle, is held in an offset holder (th). This prevents interference with the movement of the specimen stage when the device is mounted in the left side port of a Stereoscan while allowing the cutting surface of the tool to be on the axis of rotation of the shaft. The range of movement of the tool in the specimen chamber in (Y and Z) directions perpendicular to the shaft is 2.8 mm, and 10 mm in the X direction along the shaft. The micromanipulator is operated with the left hand leaving the right free to adjust the specimen stage, magnificationand stigmator controls if necessary. This is convenient and comfortable when used in conjunction with the red and green two colour stereo presentation on a large colour TV monitor situated to the right of the Stereoscan desk, but very uncomfortable when using the mirror stereoscope to view the stereo image presented on two black and white TV monitors which are sited on the left of the desk. The stereo TV display controls are adjusted for a particular working distance and accelerating voltage and need not be touched whilst operating the manipulator. The presence of the micromanipulator does not prevent the use of the long secondary electron collector, or the energy dispersive X-ray detector mounted in the left back three-quarter port: these, and other devices such as transmitted electrons detectors may be left in place. Some modifications to existing specimen stages may, however, be required. Access to the left hand side of the specimen was blocked by the side of the specimen stage ‘box’ in early Stereoscan stages: thus the side may need to be cut away. For micromanipulation purposes the specimen rigidity is not adequate with the usual plug-in type of specimen support. The specimen should, for example, be screwed to the tilting head of the specimen stage. For some applications, the sloppiness of the tilting mechanism
* Research Instruments Ltd, Cumberland Avenue, London “10, 266
Model D-10.
SEM manipulator
\+ 0
=
\
267
J. B. Pawley and A . Boy&
Fig. 2. Photograph of the micromanipulator described in the text.
Fig. 3. Chick embryo, glutaraldehyde fixed, Os04 post fixed and critical point dried, which has been dissected using a tungsten microneedle approaching from upper left. Dorsal surface at bottom. The neural groove (opening towards bottom) and notochord have been exposed by removing endoderm and mesoderm. This is a Polaroid photograph of one frame of alternate line scan mode of stereo operation taken from black and white T V monitor.
268
SEM manipulator
Fig. 4. Bone surface covered with bone forming cells (osteoblasts) prepared by glutaraldehyde fixation and critical point drying. A tungsten microneedle is being used to impale and remove individual cells in order that the orientation of these cells may be compared with that of the underlying collagen. ‘Still’ photograph from normal record CRT. Width of field is 90 pm.
is a nuisance because the specimen moves when contacted by the micromanipulator tool. We have modified a stage by removing the tilting and rotating mechanisms, leaving only X, Y and Z movements: specimen orientation must then be achieved whilst mounting the specimen. PERFORMANCE
The manipulator has been used successfully in several studies (Boyde, Jones & Pawley, 1974). While extremely rigid to forces colinear to the shaft axis, motion in the directions perpendicular to the shaft produced by forces exerted at the tip m/kg when the system is are characterized by a spring constant of 2.7 x under operating vacuum. This flexibility is due chiefly to the compression of the larger O-ring, though this is minimized by the large size of the ball and could be removed by applying heavy spring loading to the ball. However, for cutting thin sections of biological hard tissues, using a tungsten-carbide dental chisel as a tool, this was not necessary. In this case the tool was moved into the centre of the SEM field of view and then locked. The tissue was fed past the tool using the controls of a slightly modified Stereoscan specimen stage and the forces on the tool were predominantly axial. In fact, most movement of the cutting edge could be traced to flexion in the stainless steel shank of the dental chisel. This flexibility was not
269
J. B. Pawley and A. Bqde removed as it gave a useful indication of the amount of force being exerted on the tool and, thereby, reduced the possibility of damaging it. When using the joystick to move the tool against a stationary workpiece it was found that the plane chosen for this motion (YZ)had the advantage of permitting the observation of the edge during cutting as the tool was moved in a generally vertical direction, but with the disadvantage that it was hard to keep the tool in focus during this procedure. An analogue electrical readout of joystick position to provide automatic focus compensation has been considered but not yet installed. Rigidity and responsiveness were adequate up to a magnification of x 500, although practice was required to overcome the reversal in the sense of the motion caused by the action of the pivot. Also, if the point of contact was not reasonably close to the axis of the shaft some backlash and lack of rigidity could be traced to rotational motion of the pin in the slot and to bending of the wires in the connecting collar respectively. T o avoid this, the tool holder was mounted outside the column on a piece of 9-5 mm stock having a cone machined on the end and the tool was then lined up on the tip of the cone. Studies of a more precise and delicate nature have also been carried out with this apparatus. By replacing the dental tool holder with a hypodermic tube tipped with an electro-etched tungsten needle and using the manipulator only to centre the tip of this needle in the field of view, it has been possible to remove individual osteoblastic cells from the surface of bone samples by moving the same with the stage controls (Boyde, Jones & Pawley, 1974). This process was greatly aided by the real-time stereo T V viewing system incorporated in our microscope (Boyde, 1974). Once impaled on the needle tip the cells remained attached to it until they were removed by wiping them off on an adjacent piece of sample. The rectilinear motions possible with the stage controls were sufficiently sensitive to make this task quite routine. ACKNOWLEDGMENT
The development of this device was assisted by an MRC grant to Dr A. Boyde. We would also like to thank Mr R. Sampson and Mr P. Black for their assistance in fabricating it and Professor D. Picton for helping us measure the rigidity of the apparatus. Mrs Mary Bancroft provided the specimen shown in Fig. 3 and Dr Sheila J. Jones provided Fig. 4.
References Boyde, A, (1974) A Stereo-Plotting Dm'ce for SEM Micrographs and a Real Time 3-D System for the SEM. Scanning Electron Microscopy 1974. (Ed. by 0.Johari), p. 93. IIT Research Institute, Chicago, Ill. Boyde, A., Jones, S.J.& Pawley, J.B. (1974) Some Practical Applications of Real Time T V Speed Stereo SEM in Hard Tissue Research. Scanning Electron Microscow 1974 (Ed. by 0.Johari), p. 109. IIT Research Institute, Chicago, Ill. MacAlear, J.H., Germinario, L. & Fucci, R. (1971) Retinal elements revealed by SEM uyomicrodissection. Ann. Proc. Electron. Microsc. SOC.Am. 29, 446. Pawley, J.B. (1972) A dual needle piezoelectric micromanipulator for the SEM. Rew. scient. Instrum. 43-4,600. Pawley, J.B. & Nowell, J.A. (1973) Microdissection of Biological SEM Samples for Further Study in the TEM. Scanning Electron Microscopy 1973 (Ed. by 0.Johari), p. 333. IIT Research Institute, Chicago, Ill.
270
Short Technical Note A robust micromanipulator for the scanning electron microscope
by J. B. PAWLEY* and A. BOYDE,Department of Anatomy and Embryology, University College, Gower Street, London WC1E 6BT SUMMARY
A simple device for holding and moving mechanical tools in the region of a sample being viewed in the scanning electron microscope is described. The unit has a 20: 1 mechanical reduction and when fitted with a tungsten carbide dental chisel, it is sufficiently rigid to cut biological hard tissues. Alternately, when fitted with an electro-etched tungsten needle, it can be used, in conjunction with specimen stage controls, to remove individual cells from the surface of soft tissues. Examples of these applications are illustrated. INTRODUCTION
Several devices have been developed to make possible the controlled mechanical disruption being viewed in the scanning electron microscope (SEM) (MacAlear, Germinario & Fucci, 1971; Pawley, 1972; Pawley & Nowell, 1973). These have been of two general kinds. In the first, relative motion between tool and the sample surface is provided by the use of the controls normally used for positioning the sample, while in the second such relative motion is provided by movement of the tool itself. Both approaches have their advantages. The first, lacking the need for fine mechanical motions, is easier to construct and also has the added advantage that the area of greatest interest where the tool touches the sample, remains in a fixed position on the SEM display screen. On the other hand, the second approach offers a more familiar feel for intricate operations because we normally move the tool and not the workpiece in everyday life. Also, motions in directions not parallel to the axes of stage motion are more easy to perform. This paper describes a micromanipulator which embodies some aspects of both approaches. It was primarily developed to hold various tools so that the process of cutting dental tissues could be viewed in the SEM. As these materials are very hard, the rigidity of the tool-holding end of the manipulator was a major concern. Of secondary importance was the desirability of being able to make co-ordinated motions in at least two dimensions with sufficient precision to be useful at low magnifications ( x 50-400) while leaving fine relative motions to be performed by the stage motion controls.
* Present address : Department of Veterinary Anatomy, University of California, Davis, California, U.S.A. 265
J . B. Pawley and A . Boyde DESIGN
The result is shown in Fig. 1. The design utilizes as a tool support a 9.5 mm diameter stainless steel shaft (s) whose axis intersects the electron optical axis at a working distance of 8 mm. This pivots on a ball-joint made from a 26 mm ballbearing @) which is sealed to the shaft and to the outer tube (ot) by O-rings (or) and held in place by atmospheric pressure. The outer tube is soldered to a standard Stereoscan side plate (sp). Motion of the tool in the directions perpendicular to the axis of the shaft is produced by a lockable two-dimensional mechanical manipulator (yz) and utilizing a 10: 1 pivoting-level reduction* and ball bearing slides (y and z) and attached to the outer tube with a metal plate. Because of the relative sizes of the level arms on either side of the pivot point the overall mechanical reduction was 20:l in directions perpendicular to the shaft axis. The manipulator is connected to the inner tube (it) which surrounds the shaft by a special collar (rc) which uses four straight pieces of spring wire (w) to provide a small mount of flexibility in order to prevent binding as the angle between the shaft and the manipulator varies during shaft movement. The collar can be rotated in the clamp where it is attached to the manipulator permitting the tool angle to be varied and then fixed by using a locking screw (rl). Movement of the tool in the third orthogonal direction, that parallel to the shaft axis, is provided by a screw (X) which moves the shaft with respect to the inner tube. Relative rotation between these last two components is prevented by a pin sliding in a slot (ss). Atmospheric pressure on the end of the shaft provides ‘spring’ loading to remove back-lash in the screw drive. The tool (t), e.g. a dental bur or an electro-etched tungsten needle, is held in an offset holder (th). This prevents interference with the movement of the specimen stage when the device is mounted in the left side port of a Stereoscan while allowing the cutting surface of the tool to be on the axis of rotation of the shaft. The range of movement of the tool in the specimen chamber in (Y and Z) directions perpendicular to the shaft is 2.8 mm, and 10 mm in the X direction along the shaft. The micromanipulator is operated with the left hand leaving the right free to adjust the specimen stage, magnificationand stigmator controls if necessary. This is convenient and comfortable when used in conjunction with the red and green two colour stereo presentation on a large colour TV monitor situated to the right of the Stereoscan desk, but very uncomfortable when using the mirror stereoscope to view the stereo image presented on two black and white TV monitors which are sited on the left of the desk. The stereo TV display controls are adjusted for a particular working distance and accelerating voltage and need not be touched whilst operating the manipulator. The presence of the micromanipulator does not prevent the use of the long secondary electron collector, or the energy dispersive X-ray detector mounted in the left back three-quarter port: these, and other devices such as transmitted electrons detectors may be left in place. Some modifications to existing specimen stages may, however, be required. Access to the left hand side of the specimen was blocked by the side of the specimen stage ‘box’ in early Stereoscan stages: thus the side may need to be cut away. For micromanipulation purposes the specimen rigidity is not adequate with the usual plug-in type of specimen support. The specimen should, for example, be screwed to the tilting head of the specimen stage. For some applications, the sloppiness of the tilting mechanism
* Research Instruments Ltd, Cumberland Avenue, London “10, 266
Model D-10.
SEM manipulator
\+ 0
=
\
267
J. B. Pawley and A . Boy&
Fig. 2. Photograph of the micromanipulator described in the text.
Fig. 3. Chick embryo, glutaraldehyde fixed, Os04 post fixed and critical point dried, which has been dissected using a tungsten microneedle approaching from upper left. Dorsal surface at bottom. The neural groove (opening towards bottom) and notochord have been exposed by removing endoderm and mesoderm. This is a Polaroid photograph of one frame of alternate line scan mode of stereo operation taken from black and white T V monitor.
268
SEM manipulator
Fig. 4. Bone surface covered with bone forming cells (osteoblasts) prepared by glutaraldehyde fixation and critical point drying. A tungsten microneedle is being used to impale and remove individual cells in order that the orientation of these cells may be compared with that of the underlying collagen. ‘Still’ photograph from normal record CRT. Width of field is 90 pm.
is a nuisance because the specimen moves when contacted by the micromanipulator tool. We have modified a stage by removing the tilting and rotating mechanisms, leaving only X, Y and Z movements: specimen orientation must then be achieved whilst mounting the specimen. PERFORMANCE
The manipulator has been used successfully in several studies (Boyde, Jones & Pawley, 1974). While extremely rigid to forces colinear to the shaft axis, motion in the directions perpendicular to the shaft produced by forces exerted at the tip m/kg when the system is are characterized by a spring constant of 2.7 x under operating vacuum. This flexibility is due chiefly to the compression of the larger O-ring, though this is minimized by the large size of the ball and could be removed by applying heavy spring loading to the ball. However, for cutting thin sections of biological hard tissues, using a tungsten-carbide dental chisel as a tool, this was not necessary. In this case the tool was moved into the centre of the SEM field of view and then locked. The tissue was fed past the tool using the controls of a slightly modified Stereoscan specimen stage and the forces on the tool were predominantly axial. In fact, most movement of the cutting edge could be traced to flexion in the stainless steel shank of the dental chisel. This flexibility was not
269
J. B. Pawley and A. Bqde removed as it gave a useful indication of the amount of force being exerted on the tool and, thereby, reduced the possibility of damaging it. When using the joystick to move the tool against a stationary workpiece it was found that the plane chosen for this motion (YZ)had the advantage of permitting the observation of the edge during cutting as the tool was moved in a generally vertical direction, but with the disadvantage that it was hard to keep the tool in focus during this procedure. An analogue electrical readout of joystick position to provide automatic focus compensation has been considered but not yet installed. Rigidity and responsiveness were adequate up to a magnification of x 500, although practice was required to overcome the reversal in the sense of the motion caused by the action of the pivot. Also, if the point of contact was not reasonably close to the axis of the shaft some backlash and lack of rigidity could be traced to rotational motion of the pin in the slot and to bending of the wires in the connecting collar respectively. T o avoid this, the tool holder was mounted outside the column on a piece of 9-5 mm stock having a cone machined on the end and the tool was then lined up on the tip of the cone. Studies of a more precise and delicate nature have also been carried out with this apparatus. By replacing the dental tool holder with a hypodermic tube tipped with an electro-etched tungsten needle and using the manipulator only to centre the tip of this needle in the field of view, it has been possible to remove individual osteoblastic cells from the surface of bone samples by moving the same with the stage controls (Boyde, Jones & Pawley, 1974). This process was greatly aided by the real-time stereo T V viewing system incorporated in our microscope (Boyde, 1974). Once impaled on the needle tip the cells remained attached to it until they were removed by wiping them off on an adjacent piece of sample. The rectilinear motions possible with the stage controls were sufficiently sensitive to make this task quite routine. ACKNOWLEDGMENT
The development of this device was assisted by an MRC grant to Dr A. Boyde. We would also like to thank Mr R. Sampson and Mr P. Black for their assistance in fabricating it and Professor D. Picton for helping us measure the rigidity of the apparatus. Mrs Mary Bancroft provided the specimen shown in Fig. 3 and Dr Sheila J. Jones provided Fig. 4.
References Boyde, A, (1974) A Stereo-Plotting Dm'ce for SEM Micrographs and a Real Time 3-D System for the SEM. Scanning Electron Microscopy 1974. (Ed. by 0.Johari), p. 93. IIT Research Institute, Chicago, Ill. Boyde, A., Jones, S.J.& Pawley, J.B. (1974) Some Practical Applications of Real Time T V Speed Stereo SEM in Hard Tissue Research. Scanning Electron Microscow 1974 (Ed. by 0.Johari), p. 109. IIT Research Institute, Chicago, Ill. MacAlear, J.H., Germinario, L. & Fucci, R. (1971) Retinal elements revealed by SEM uyomicrodissection. Ann. Proc. Electron. Microsc. SOC.Am. 29, 446. Pawley, J.B. (1972) A dual needle piezoelectric micromanipulator for the SEM. Rew. scient. Instrum. 43-4,600. Pawley, J.B. & Nowell, J.A. (1973) Microdissection of Biological SEM Samples for Further Study in the TEM. Scanning Electron Microscopy 1973 (Ed. by 0.Johari), p. 333. IIT Research Institute, Chicago, Ill.
270
