Use of an Automated Microscope Platform for Protein and Small-Molecule Crystal Harvesting

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Please check out our Laboratory Microscopes section for more information or to find manufacturers that sell these products.

A standard laboratory microscope is a necessary tool in almost every laboratory in the world. While electron, fluorescence, confocal, and atomic force microscopes (AFMs) are gaining in popularity, the standard laboratory microscope continues to be a basic requirement. It is hard to imagine a laboratory without one.

The standard microscope has gone through many changes over the last few decades, including the addition of digital imaging via a standard trinocular configuration with a third viewing port and various types of automation. These include automated slide handling, autofocus, powered zoom, and an automated aperture. While individual features have been automated, very few entire procedures have been automated.

Figure 1 - The Harvester-ST.

Figure 2 - The Harvester joystick.

To automate or even semiautomate many common experiments currently performed under a microscope would involve many of the components mentioned above, in addition to a patchwork of XYZ motion control devices, manual micromanipulators, handheld pipettors, robotic software, imaging software, temperature control stages, shakers, vortexers, etc. The SAMI series integrated imaging system (Station for Automated Microscopy and Imaging), including the Harvester (FMP Products, Inc., Greenwich, CT) automates many standard laboratory procedures that require use of the components mentioned above (see Figures 1–4).

Figure 4 - The Harvester-3D.

Figure 3 - System manipulator.

Automated crystal harvesting

One of the first laboratory procedures to be automated was crystal harvesting (also known as crystal mounting or looping a crystal), a brief description of which follows. The harvesting of crystals can be very complicated. Crystals can range from several microns to several hundred microns in diameter. Some are almost entirely made up of liquid (protein crystals). These tend to be extremely fragile and are difficult to manipulate because they often stick to surfaces (especially certain types of plastic), group together, move within the drop as the user tries to harvest them, dry up (crash out) if too much time is taken to retrieve them from the drop, and so on. Small-molecule crystals, such as salt, tend to be more stable (but not always).

To begin harvesting protein or small- molecule crystals, a slide or plate containing the crystals is placed under a microscope, usually in a drop of mother liquor (from <1 μL to greater than 10 μL) or oil, or sometimes they are placed dry on the microscope slide. In the case of protein crystals, the researcher uses a small “loop” (10–200 μm), normally made of plastic and attached to a small handle about the size of a pencil, and tries to manually capture the crystal using the surface tension of the liquid suspended within the loop. For smallmolecule crystals, the researcher can either mount the crystal in a loop containing oil for surface tension support, or use UV-curable glue on the end of a pin to harvest the crystal. Once the crystal is mounted, it is flash-cooled to stabilize it and prevent it from degenerating. At this point, the crystal is either analyzed in-house or placed in a synchrotron—a cyclic particle accelerator in which the magnetic field (to turn the particles so that they circulate) and the electric field (to accelerate the particles) are carefully synchronized with the traveling particle beam—for further investigation.

Challenges of manual harvesting

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.

Automated microscope platform

The basic device used was the Harvester-3D automated microscope platform with dual micromanipulators. This robotic imaging and sample manipulation platform offers automatic plate scanning, 3-D imaging, crystal harvesting, and a fully programmable macro language and a complete assortment of tools. The macro language includes commands that instruct the system to carry out its operations unattended. Once initiated, macros can operate the microscope for minutes, hours, or even days (i.e., time-lapse studies).

For this application, the integrated on-board options and software included:

  • A rotational microscope capable of magnifications from 50× to 400× (Hirox, River Edge, NJ)
  • Autozoom and autofocus (FMP)
  • A stage with automated XYZ capabilities (FMP) and multiple axes of freedom
  • Control for up to 21 motorized devices (FMP)
  • Dual multiple-axis submicron manipulators (up to four) (FMP) on moveable platforms
  • Dual metal-halide light sources (150 W) (Welch Allyn, Skaneateles Falls, NY)
  • Dual fiber optic pathways (simultaneous brightfield and darkfield lighting)
  • UXGA digital scientific camera (Lumenera Corp., Ottawa, Ontario, Canada)
  • MiTeGen ( I tha c a , NY) mount s for looping and manipulating crystals or the Crystal Catcher (Kyodo International, Tokyo, Japan) (virtually any loop or tool will work with the system)
  • Image Automation V3.65 software for image capture and robotic control (FMP).

System operation

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.

  • Step 4—Loop to step 3 until finished (note: macros have “if/then/else” capabilities).

The entire process of writing the macro to accomplish this task took about 2 hr, with a total of 10 steps. Although the system is capable of controlling up to 21 axes or devices simultaneously, thi s appl icat ion requi red only 11. Other experiments and procedures that can be successfully programmed on the device include autorotation and global 3-D imaging with Z-stacked images, automatic dispensing of reagents in a 96-well plate with mixing and automated image capture, automatic plate scanning with 3-D rotational imaging and image analysis, automated inspection of parts, and autohandling and imaging and storage.


With the ability to control multiple devices, the SAMI series of automated microscopes offers researchers a powerful tool for designing applications or performing specific tasks (such as harvesting crystals) in one convenient package. The integrated macro language allows microscopists and researchers to create powerful, automated solutions to imaging and sample manipulation problems.

Please check out our Laboratory Microscopes section for more information or to find manufacturers that sell these products.

Mr. Friedlander is President, FMP Products, Inc., 100 Melrose Ave., Ste. 206, Greenwich, CT 06830, U.S.A.; tel.: 914-939-3014; fax: 866- 226-1795; e-mail: [email protected].