The Protein Crystal Mounting Project

The goal of the Protein Crystal Mounting project is to produce a microrobotic system capable of autonomously mounting protein crystals on a tool for the purpose of X-ray data collection. The system uses visual feedback from a camera mounted on a microscope to control a micromanipulator which has the mounting tool attached as its end-effector. For use with our robotic system, we have developed unique new tools, called miscroshovels, which are designed to address certain limitations of the traditionally used by crystallographers cryogenic loops and capillaries.

The motivation for this project, along with some background information, is described separately.

A coverslip (such as of a 24-well Linbro plate) containing a droplet with protein crystals is placed under the microscope. The operator uses a tool to catch and pick up a target crystal looking through the microscope. The most-commonly used tool for mounting is the cryogenic loop (e.g. ones made by Hampton Research), though glass capillaries are also used. The crystal is then quickly cryoprotected, frozen and stored for future data collection on an X-ray beamline.

The video on the right is an example of what the task entails --- the loop in this video was installed on a micropositiner which was teleoperated. In reality, the task may be further complicated by the limitted time for operation, "skins" forming on the surface of the droplet and crystals adhering to the coverslip.

Accuracy and speed are critical as the crystals are fragile and very sensitive to environmental changes. Dehydration quickly leads to crystal quality degradation.

Crystal mounting is a term thas has been used to refer to both the task of picking up a protein crystal (a.k.a crystal harvesting) and the task of placing the tool with the crystal already on it on the beamline. In our work, we use the term to mean the former, and the latter we refer to as beamline mounting to avoid confusion.

We have designed and assembled a micro-robotic system for protein crystal manipulation, which we use for our research and experiments. The system uses our own custom tools, called microshovels, which we designed and fabricated using MEMS technology.

The system operates as follows: The user places the coverslip with the crystals under the microscope, starts the program and selects a target crystal. After that, the program proceeds in three phases: descent, approach and pickup.

During the descent phase, the miscroshovel is lowered into the droplet all the way down to the surface of the coverslip. The main difficulty in this phase is to detect when the correct depth is achieved. To do this we utilize the rigidity of the silicon. The systems continues its downward motion in large steps (100-200um) until the tool touches the plastic coverslip. At the time of contact between the tool and the coverslip, due to the rigidity of the silicon, the coverslips slides horizontally under the force. To accentuate the horizontal component, we use a zig-zag descending motion. We detect the motion using optical flow methods on the video feed from the camera.

After the tool is in focus, we control its horizontal movement to slide it under a target protein crystal for pickup. The approach phase consists of visual servoing of the tool toward the crystal such that the tool is positioned immediately before the crystal but there is no contact between them. Both the tool and the crystal are tracked using region trackers on the video stream. The tracker for the tool is initialized automatically, by briefly withdrawing the tool from the field of view and using image differences to locate its position. The crystal is selected manually, as visual categorization and evalution of the quality of different crystals is beyond the scope of our work for now.

Finally, when the tool and the crystal are aligned, the pickup phase consists of rapidly sliding the tool under the crystal and lifting it up. The video on the right shows the entire 3-phase process, peformed automatically by the system. After the successful mounting, the refocusing, the repositioning of the light and the tool were performed manually for a better view of the crystal on the tool (these are not part of the regular procedure).