CrystalDirect Automatic Crystal Harvester

This video presents CrystalDirect®, a fully-automated crystal harvester robot working in a high throughput crystallography environment.
CrystalDirect® is a co-development of the European Molecular Biology Laboratory (EMBL) and Arinax Scientific Instrumentation.

High throughput harvesting process

CrystalDirect® uses EMBL well plates which are commercially available from MiTeGen (CrystalDirectTM plates). These plates are fully compatible with standard 96-well plates. They are composed of a plastic frame and a thin membrane on which the samples are deposited (“hanging drop” type). The CrystalDirect® robot cuts the membrane around one or more than one selected crystals by means of laser photoablation. (Cipriani et al.,2012)

Thereafter the crystal support is fixed on a SPINE sample pin and immediately cryo-cooled.

Thanks to this laser photoablation technology, CrystalDirect® gives also the possibility to separate nested crystals by laser cutting from crystal agglomerates (crystal surgery). Protein crystals harvested by this technique do not get deteriorated, neither does their diffraction power as could be demonstrated by numerous experiments. (Zander et al.,2016)

CrystalDirect® is a fully-automated system designed for high throughput crystal harvesting that fits perfectly to modern crystallization platforms: CrystalDirect® needs about 1 minute per harvest and reaches this performance with a success rate which is superior to 99 %.

Intended for highly automated workflow, CrystalDirect® is compatible with a large variety of sample changer robots and especially with the Flex ED 6-axis Robot, developed by the EMBL.

Multiple selectable harvesting methods

CrystalDirect® offers a large amount of cut shapes for the laser photoablation process.  The different shapes adapt to different protein crystal distributions and orientations on the native membrane. Consequently, CrystalDirect® can select and harvest single crystals, batches of crystals, micro-crystals and single needles from clusters.

From the left to the right: single crystal selection, multiple crystal selection, single needle crystal selection

Examples of cut shapes for automated crystal harvesting which are integrated in the software menu of CrystalDirect

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Integrated cryo-cooling process

CrystalDirect® features an integrated cryo-cooling process. Just after each harvest, the system automatically moves the protein crystal into a gaseous stream of 100 K cold nitrogen.

The cryo-cooled sample can be transferred into a Dewar either manually or by means of a sample changer robot.

Cryo-protection by liquid removal

CrystalDirect® enables the removal of the mother liquor surrounding the protein crystals. In this way, the risk of mechanical stress to the crystal during the cryo-cooling process is strongly reduced. As a result, in most cases it isn’t necessary to add cryo-protectant (Pellegrini et al., 2011).

This automatic liquid removal process has a high level of quality (Zander, U., et al., 2016). In addition, the operating time and the repeatability are highly optimized compared to the manual approach. 

In situ soaking

The combination of the CrystalDirect® harvesting robot with CrystalDirect™ plates allows liquids to be added to the protein crystal with reduced risk of mechanical or chemical stress: a micro aperture is cut in the plate membrane by laser photoablation.

Then, a drop of liquid is placed on the aperture and, in this way, the added liquid gradually diffuses into the mother liquor. This gentle method of in situ crystal soaking reduces the risk of chemical stress and eliminates any risk of mechanical stress which comes with crystal manipulation.

Web based management

CrystalDirect® is aimed to be integrated into a fully automated environment, ranging from pre-crystallization steps (sample management) to diffraction data analysis. Such an environment is generally handled via specialized management software. CrystalDirect® is able to work hand-in-hand with such crystallization management systems.

The EMBL has developed a web based application called CRIMS (CRystallization Information Management System). CRIMS takes in charge data management of the entire crystallization line, giving web functionalities for each step:

Sample Management

CRIMS allows recording all the data related to the samples (name, type, concentration and others), designing crystallization experiments and automatic tracking of all experimental parameters (crystallization conditions, temperature, etc.) for each well. At any time users can access to these data and modify them. CRIMS links these data with the next step, the crystallization screening.

Crystallization screening

CRIMS can collect pictures generated by Crystal Farms (e.g. Rock imagers). The operator and users can inspect and score all of these pictures for each crystallization well. This “image viewing & scoring” function allows the expert user to analyses and annotate the results for each well.

Automated protein crystal harvesting – integration with CrystalDirect®

The EMBL developed the automated protein crystal harvester CrystalDirect® to take full advantage of the features offered by CRIMS. Based on the crystallization screening results the expert user can select the crystals to be harvested and defines the cut shape and its orientation for each harvesting process. Then, CRIMS transfers the information to CrystalDirect® which will perform the harvest as set.

CrystalDirect® cryo-protects harvested crystals, takes pictures of the cryo-cooled samples and transfers these images to CRIMS. The expert user can check these images by means of CRIMS and hence, gets visual feedback on the success of the harvesting process and the final sample quality.

Following the harvest, CrystalDirect® makes sample pins available to a sample changer robot which stores the cryo-protected crystals in liquid nitrogen.

Integration with Data Collection

Once the collection of diffraction data is finished, CRIMS retrieves a summary of the data quality from the synchrotron’s diffraction database, e.g. ISPyB.

CRIMS is a development of the EMBL Grenoble – HTX laboratory (Márquez Team)

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Technical specifications

Harvesting time 1 crystal/min
Success rate >99 %
Laser cutting No heat transfer
Micrometric precision
Multiple cut profiles
(pre- & user-defined)
Crystal soaking process 500 µL reservoirs
film piercing: photo ablation
User interface Windows / JAVA
Web based crystal selection CRIMS, customizable
Multi-device servers TINE, TANGO, EPICS
Sample changer robot Robot hardware & software integration
230 VAC; 50 Hz <3680 W
110 VAC; 60 Hz <3680 W
Dimensions (X,Y,Z)
1.0 x 1.0 x  2.3 m3

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