Protein Crystal Mounting
Fig.1: A crystal mounted on a microshovel
The Protein Crystal Mounting project addresses the need in the Protein
Crystallography community for high-throughput (HTP) equipment which will help
improve the execution of a specialized task, called crystal mounting.
The goal of this project is to produce a microrobotic system capable of
performing the task autonomously, quickly and robustly. We have built
a robotic system for crystal mounting which relies on visual feedback
from a camera looking through a microscope to control a micromanipulator
with the tool used for mounting attached as its end-effector.
The motivation for this project, along with some background
information, is described
Fig.2: Crystal mounting tools
Video: A crystal mounting example (8MB)
Fig.3: Crystal mounting in the HTP pipeline
Crystal mounting is simply described as the transfer of a selected
protein crystal from its growth solution into a suitable mounting tool for
data collection on a synchrotron. The task begins by placing a coverslip (usually
a 21mm x 21mm square plastic slide, such as the ones used for 24-well Linbro plates)
containing a droplet with protein crystals is placed under the microscope. The
technician, looking through the microscope, uses a tool to catch and pick up the
selected target crystal. The crystal is then quickly cryoprotected, frozen and
stored for future data collection on an X-ray beamline.
The most-commonly used tool for mounting is the cryogenic loop (e.g. ones made by
Hampton Research) though glass capillaries are also used. Recently, newly developed
tools have been introduced, such as the micromounts manufactured by MiTeGen and our
own microshovels. Fig.2 gives a visual
comparison of the loop, the micromount and the microshovel.
The task is currently performed by skilled technicians. Mounting a crystal in
a loop manually requires time, patience and excellent motor skills. Accuracy
and speed are critical as the crystals are fragile and very sensitive to
environmental changes. Dehydration quickly leads to crystal quality degradation.
The video in the side panel is an example of what the task entails --- the loop in
this video was installed on a micropositioner which was teleoperated. In reality,
the task may be further complicated by the limited time for operation, "skins"
forming on the surface of the droplet and crystals adhering to the coverslip.
Crystal mounting is a term that 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 (Fig.3).
System Design And Operation
We have designed and assembled a
protein crystal manipulation, which we use for our research and
experiments. An earlier version of the system successfully demonstrated
the use of our microshovels
for autonomous mounting. The current implementation relies on a two-stage
approach, where one tool (a glass pipette) is used to pick up the
crystal from the incubation drop and transfer it to another tool (a
micromount or a microshovel), which holds the crystal during X-ray data
Fig.4: Crystal mounting setup
Fig.5: Control system block diagram
Video: Automatic crystal mounting (1MB)
This method is beneficial for several reasons. First, it makes catching the crystal more
robust, since the microinjector can adjust the amount of suction it applies via the pipette,
compared to the methods that use a loop, a micromount or a microshovel directly, which rely
on surface tension to hold the crystal. Second, the pipette aspirates some of the mother
liquid along with the crystal and provides an enclosed natural environment for the crystal
which protects it from dehydration when it is extracted from the drop; the crystal never
gets exposed to the damaging effects of room conditions. Thirdly, this approach naturally
combines two steps of the pipeline: crystal mounting and cryoprotection. And finally,
problems typically associated with the use of pipettes in cryoprotection (e.g. difficulty
in flash freezing because of lack of sufficient exposure) and data collection (e.g. excessive
amount of liquid around the crystal) are avoided by transferring the crystal to a more
appropriate tool for the purpose.
The crystal mounting procedure starts with the placement of the necessary
tools and objects in the work space (Fig.4). First, a microbridge with cryoprotector
is placed at its designated location on the tray. Next, a micromount is installed
on the fixture to the right and is positioned adequately so it is immersed in the
cryprotector at an angle of approximately 45 degrees and is ready to receive the
crystal. Finally, the user places a coverslip with the droplet containing the
protein crystals on the microscope tray such that they are in the field of view.
The user starts the program and specifies which crystal (among the possibly many in
the drop) is to be mounted. After that, the system proceeds autonomously: It
immerses the pipette into the drop, approaches the crystal, aspirates the crystal,
transitions from the drop on the coverslip to the cryoprotector in the microbridge
and deposits the crystal in the mounting tool. Some of these steps are performed in
open-loop fashion because the system is calibrated for the locations and dimensions
of the relevant objects and the system actuators meet the requirement for positioning
accuracy. The aspiration of the crystal, however, is an example of a step that
crucially depends on reliable sensory feedback. To determine the location of the crystal
and detect when the crystal is inside the pipette, we use region trackers applied to the
visual feed from the camera. A control loop tracks the motion of the crystal as it is
drawn into the pipette and adjusts the suction applied by the microinjector accordingly
until the crystal is safely inside. A block diagram of the control algorithm is shown
in Fig.5. A video of the system in operation is shown in the side panel.
Two-Stage Robotic Crystal Mounting of Protein Crystals
for X-Ray Data Collection
In Proc. of the IEEE Int. Conf. on Automation Science and Engineering
(CASE'08), Arlington, VA, August 2008, pp.1019-1024 (paper & poster).
(with Peter K. Allen)
Visually-Guided Protein Crystal Manipulation Using
Micromachined Silicon Tools
In Proc. of the IEEE/RSJ Int. Conf. on Intelligent Robots and Systems
(IROS'04), Sendai, Japan, September 2004, pp.236-241.
(with Peter K. Allen and William Edstrom)
Microrobotic Crystal Mounting Using Computer Vision
Microrobotics for Biomanipulation Workshop, IEEE/RSJ Int. Conf. on
Intelligent Robots and Systems (IROS'03), Las Vegas, NV, October 2003.
(with Peter K. Allen and Youcef Mezouar)
Visual Servoed Micropositioning for Protein
In Proc. of the IEEE/RSJ Int. Conf. on Intelligent Robots and Systems
(IROS'02), Lausanne, Switzerland, October 2002.
(by Youcef Mezouar and Peter Allen)