3.1. Grid handling
A Yamaha YK250X SCARA robot is employed for loading EM grids from a 96-well tray into the sample holder (). The robot has four motors, three of which generate rotation about the vertical (z) axis and one of which generates linear movement along this axis. The rotational movements are combined to generate linear movement of the grid handling probe in the x-y plane, which is used to move between the grid tray and the sample holder. The precision of these movements are ±0.01mm for translation and ±0.01° for rotation. The SCARA robot has a small footprint (~4000 cm2) and is mounted on a table ~1 m above and behind the microscope console. This table also accommodates the EM sample holder and the 96-well EM grid tray, which are both within the limited range of motion afforded by the SCARA robot design.
Fig. 2 Loading EM grids. (A) A SCARA robot has been implemented for handling individual EM grids. (B) The SCARA robot is fitted with a vacuum probe to pick up grids using three nozzles positioned around the rim of the grid. (C) Grids are stored in an anodized (more ...)
The EM grid tray () was designed with standard SBS dimensions with 96-wells that are 0.9mm deep and 3.1mm in diameter. This diameter is only slightly larger than the standard EM grid (3.05mm), thus ensuring that the grids remain centered. The trays have an overall height of 10mm and were manufactured from aluminum using a programmable CNC mill and were black anodized. Since the trays are relatively inexpensive, many copies were made to allow storage of screens for potential reevaluation at a later date. The trays are securely held by a spring-loaded clamp to ensure that they are precisely positioned relative to the SCARA robot.
Different probes were evaluated for transferring grids from the storage tray to the sample holder. Forceps are commonly used for manual manipulation of EM grids, but were rejected as difficult to implement for a robot probe. As an alternative, we designed a vacuum probe consisting of three nozzles that are mounted vertically at the tip of the robot arm (). The nozzles have an inner and outer diameter of 0.25 and 0.5mm, respectively and contact the rim at the perimeter of the grid, thus minimizing damage to the carbon film. The vacuum was generated using the Bernoulli effect, for which we used oil-free compressed air to feed a vacuum valve (Anver Corp, Hudson MA) that was mounted under the robot stand and connected to the nozzles through tygon tubing. Suction is turned on by simply applying voltage to the valve and the resulting vacuum pressure was controlled by the input pressure (60 psi) to optimize grid pickup. We found that this configuration minimizes grid deformation that might otherwise cause mishandling or misplacement of individual grids. A single nozzle was also effective in lifting the grids but, if applied to the center of the grid, it routinely damaged the carbon film and, if applied to the rim, the grids tended to rotate thus becoming incorrectly positioned for release onto the holder. Commercial rubber suction cups were also evaluated, but these tended to generate static electricity and furthermore contacted a large region of the grid, potentially causing widespread damage to the carbon film. Using the vacuum probe, the SCARA robot has a 98% success rate in transferring grids to the holder. The remaining 2% never get picked up from the grid tray, presumably due to grid deformation. The nozzle occasionally becomes magnetized and therefore fails to release the grids onto the holder, a situation which is quickly remedied with a demagnetizer.
3.2. Sample holder
The tip of the standard EM sample holder was redesigned to accommodate operation by the SCARA robot (). In particular, loading an EM grid into the high stability holders from JEOL requires manipulation of a delicate arm that is held in place by multiple fine screws. The standard holder for the JEOL 1230 microscope features a quick-change clamp that must be pulled down with significant force to either secure or release the grid. The unclamping is unpredictable and sometimes leads to the EM grid jumping out of position or out of the holder all together; once released, the clamp has considerable side-to-side play and is highly mobile. Fortunately, the entire tip is fastened to the arm of the sample holder by three screws and can be readily exchanged with alternative tips.
Fig. 3 Modifications to the tip and the handle of the standard EM holder. (A) The holder is shown resting in a custom mount that serves to define its location and orientation relative to the SCARA and Cartesian robots. The original plastic holder was replaced (more ...)
The new tip was milled from brass and equipped with a spring-loaded cam that can be raised and lowered in a controlled way (). This clamp has a 90° range of motion, from vertical (open) to horizontal (closed), with very little side-to-side play. The distal end of the clamp - furthest from the handle - extends beyond the end of the tip by 5mm, thus providing a handle for raising and lowering the clamp. This extension is engaged by an L-shaped finger attached to the probe of the SCARA robot next to the vacuum nozzle (). The cam-shaped hinge is loaded with a spring such that it comes to rest at either the fully open or fully closed position and applies pressure that holds the grid firmly in place.
To ensure a reproducible position of the EM sample holder relative to the two robots, a custom-made mount was constructed (). A precise orientation of the holder is maintained by a pin on the mount that inserts into a hole milled into the bottom of the holder handle.
In an attempt to verify the presence of an EM grid both in the 96-well tray and in the sample holder during transfer by the SCARA robot, a small laser was mounted on the robot probe. However, the reflected signal from the Ni-grids proved to be unreliable, due both to lack of flatness and to discoloration from carbon coating and sample staining. Instead, we decided to rely on the initial low magnification images from the EM to determine if a grid had been successfully loaded into the holder. Given the high success rate of the SCARA robot in transferring grids, this is a rare event. Once the clamp has been lowered, the grids remain securely fastened and we have not experienced any loss during transfer of the holder into the microscope.
3.3. Sample Insertion
Once the SCARA robot loads an EM grid onto the tip of the sample holder, a four-axis Yamaha MXY-x Cartesian robot picks up the holder and transfers it to the electron microscope (). A pneumatic, three-fingered gripper (P5G-HPC-320, Parker Hannifin Corp, Cleveland OH) is used to grasp the handle of the specimen holder (). The Cartesian robot then carries the holder to the entrance of the airlock using three motors to generate linear movements along the x, y, and z axes. These motors have adjustable torque, a maximum speed of 1200mm/s, and an accuracy of 0.01mm. The gripper is attached to a rotational motor, which enables manipulation of the holder through the airlock.
Fig. 4 A Cartesian robot is used to move the sample holder to the microscope and insert it through the goniometer. This robot provides three orthogonal axes for linear motion and a three-fingered gripper is mounted on a rotation stage. (A) Robot prior to picking (more ...)
The maximum torque allowed for these four motors was restricted in order to minimize potential damage to the microscope as well as the consequences of accidental contact with foreign objects. Each motor was characterized by a minimal torque required to overcome its innate, internal resistance; lower torques produced chatter or resulted in no movement at all. After empirically establishing the threshold for each motor, the torque was increased slightly above this threshold to provide reliable movement. Rotation through the airlock and removal of the holder from the column required somewhat higher torque than other movements, but in all cases motion can be easily stopped using one or two fingers (i.e., <5N of force). The iRobot program monitors each movement and if the endpoint does not correspond to the expected position, then it assumes that some impediment has been encountered and all further movements are immediately halted.
Alignment of the Cartesian robot with the axis of the microscope goniometer is critical. Improper alignment can result in damage to the microscope or to the holder, especially during rotation in the airlock. Specifically, a guide pin on the holder engages a groove on the airlock, thus opening relevant valves and gaining access to the column. As a first step in the alignment process, the Cartesian robot was mounted such that its axis of insertion was roughly parallel to the goniometer axis. However, mechanical failure of an optical switch operated by the guide pin alerted us to an angular misalignment between the insertion angle of the Cartesian robot and the goniometer. Unfortunately, neither the linear x-y-z motors nor the on-axis rotation stage allowed adjustment of the entrance angle. Nevertheless, the goniometer itself provided a mechanism for adjusting its axis of insertion relative to the robot. Specifically, translation of the EM grid inside the microscope along y and z axes is effected not by linear motions, but by slight rotations of the holder (the x axis corresponds to the rod axis and z is vertical). The pivot point for these rotations is relatively close to the EM grid, such that a small (1 mm) translation of the grid results in a much larger (5 mm) movement of the handle. Thus, moving the holder in the microscope in y and z produced different angles of the sample rod relative to the fixed coordinate system of the robot. The y axis has a larger range and required more adjustment than the z axis, since both the goniometer and the robot were mounted close to level. Translation along the rod axis (x) is in fact linear and therefore does not affect the entrance angle.
To establish an optimal alignment, the sample holder was inserted into the microscope and the unclenched grippers were positioned around the handle. The run-out was then measured as the grippers were rotated around the handle. If necessary, the holder was moved within the goniometer to a new position (i.e., a different angle) and the Cartesian robot was translated to the new position adopted by the handle. This procedure was iterated until an optimal goniometer “home” position was established, where axis of the Cartesian robot was parallel to the entrance cylinder of the goniometer. During this process, we found that the axis of the rotation stage on the Cartesian robot needed adjustment to be precisely parallel to the direction of insertion, since the robot specifications were rather lax in this regard. Also, in order to maintain the accuracy of the holder orientation, we replaced the original hollow, plastic handle of the EM holder with a solid aluminum handle, thus preventing deformation of the original hollow plastic handle by the gripper. Although the diameter of this handle was arbitrary, it was important to ensure that it was concentric with the sample holder rod and tip, so that rotations in the airlock did not give rise to displacements of the sample holder rod. Overall, we estimate a margin of error for this alignment to be about ±0.5mm. All these considerations are moot when it comes to manual insertion, because an operator does not rigidly grip the holder at a specific location or angle relative to the goniometer.
The reproducibility of this alignment requires that the microscope remain in a defined position relative to the robot. The routine use of anti-vibration mounts for electron microscopes is a potential problem in this regard, especially with air mounts that allow substantial column movement and have the potential to get out of balance, e.g., due to dirty air valves. The rubber mounts employed by the JEOL 1230 are much stiffer and appear so far to return the column to a reproducible position. However, we mounted a single small laser onto the microscope column and marked the position of its beam on the wall of the room to provide a long-term, visual monitor of the position of the column. If column movement were to become a problem, one could mount a laser on the probe and an optical sensor on the microscope; a feedback system could then be established to help the Cartesian robot home in on the optimal position prior to insertion of the holder. With very compliant anti-vibration mounts, it is conceivable that the force of insertion/removal would push the column out of alignment, in which case it would be advisable to take measures to stiffen the mounts or minimize the force used for insertion and removal.