Minggu, 06 November 2011


Crystal manipulation and harvesting have been done manually since the science of crystallography began. While the looping of larger crystals comes easily for some people, others never seem to get the knack. Some of the problems with manually harvesting crystals (both small and macro) include:
Crystals are being harvested at earlier and earlier points in their growth cycle, sometimes when they are as small as 2–5 μm. As the crystals get smaller, the ability to manually harvest them becomes more difficult.
Manual harvesting may damage the crystals due to the user’s inability to precisely control the looping tools.
When harvesting manually, higher levels of magnification required for small crystals are difficult to use because the user’s hand motions (i.e., shaking) are intensified, distorting vision and thereby affecting the ability to accurately harvest.
It is difficult to choose a specific crystal to loop since manual looping disturbs the entire drop and any other crystals that are present in that drop
Manual harvesting of crystals is done with one loop; the use of two loops simultaneously to capture crystals is rarely done by hand.

Typically, the user places a coverslip or another vessel containing the crystals under the microscope. Because time is usually limited (especially in the case of protein crystals), the user presses a single key, and the system positions the loop(s), crystals, and microscope in preparation for harvesting crystals. (Note: Sometimes a small amount of Paratone-N oil [HamptonResearch, Aliso Viejo, CA] is used to delay the dehydration of the drop holding the crystals to allow more time for harvesting.)

If the user has already selected the crystal to harvest, he/she can choose that crystal by clicking on it with the mouse. Once the desired crystal is selected, the user can harvest it manually or invoke a macro, which will harvest it automatically based on methods and training established by the user. Although several off-the-shelf macros for crystal harvesting are included with the system, the user can program in his/her own style of harvesting, with the system mimicking the user’s every move. The programmable macros control all devices available in the automated system (XYZ stage, micromanipulators, zoom, focus, etc.), as well as all software functions such as image analysis, image capture, video capture, and Z-stacked imaging.

Because the Harvester-3D can be operated remotely, it is well suited for harvesting oxygen-sensitive crystals within a glove box and offers various other benefits when used in an oxygen-free environment. For example, with its precise joystick operation, users do not have to work with gloved hands, which is a tedious, tiring, and time-consuming method of harvesting crystals.

The micromanipulators can hold either traditional loops (MiTeGen) or other types of harvesting devices, such as the Crystal Catcher. The Crystal Catcher can be programmed to automatically harvest both protein and small-molecule crystals using its polymer-based adhesive technology, and the penlike device mounts easily onto the micromanipulators.

A typical macro for harvesting crystals is as follows:
Step 1—Move stage to harvesting position. This brings the XYZ stage into position, autofocuses the microscope, and moves the micromanipulators with loops into position.
Step 2—Mark crystal(s) for harvesting. This highlights all of the crystals to be harvested (either automatically or manually).
Step 3—Begin harvesting. Depending on whether small- or macromolecule crystals are being harvested, the procedure is slightly different. For the purposes of this discussion, we will assume small-molecule harvesting is being done using UV-curable glue to attach the crystals to the mounts.

In turn, the system will:

a) Move to the UV glue position

b) Pick up a small amount of UV glue (amount predetermined by the user)

c) Move to the first crystal to be harvested

d) Pick up the first crystal using XYZ coordinates generated when the user marked the crystal (step 2)

e) Move to UV curing light for 20+ sec to harden the glue. The user can move the micromanipulator to neutral position and pause so that he or she (or the automated arm [i.e., Hitachi, Tokyo, Japan]) can remove the harvested crystal and replace the holder for the next crystal to be harvested.

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