Protein Crystal Growth Monitoring by Digital Holographic Microscope for the International Space Station-3 (PromISS-3) - 05.24.17

Overview | Description | Applications | Operations | Results | Publications | Imagery

ISS Science for Everyone

Science Objectives for Everyone
The PromISS-3 investigation includes a microscope that will allow for the visualization of the protein crystal growth process. Protein crystals are grown in microgravity to visualize proteins to aid in the development of new drugs to fight diseases. Studying the processes by which these crystals grow in microgravity will help scientist better understand the role of the proteins in diseases.
Science Results for Everyone
This investigation grew six proteins in weightlessness, demonstrating that the counter-diffusion growth method is useful for producing higher-quality crystals and for obtaining different forms with improved properties. More than 100 datasets collected from triose phosphate isomerase (TIM) crystals show a clear effect of diffusion (movement of particles from high to low concentration) and higher crystal perfection in an environment without fluid movement. For other proteins, such as cablys3*lysozyme (an antibody), diffusion had a negligible effect. These experiments show that protein crystallization is only one process in a very complex Landscape of phase behavior of protein at high concentrations.

The following content was provided by Juan Manuel Garcia-Ruiz, Ph.D., Fermin Otalora Munoz, Ingrid Zegers, Ph.D., and is maintained in a database by the ISS Program Science Office.
Information provided courtesy of the Erasmus Experiment Archive.
Experiment Details

OpNom:

Principal Investigator(s)
Juan Manuel Garcia-Ruiz, Ph.D., University of Granada, Granada, Spain
Fermin Otalora Munoz, University of Granada, Granada, Spain
Ingrid Zegers, Ph.D., Free University, Brussels, Belgium

Co-Investigator(s)/Collaborator(s)
James Lutsko, Free University, Brussels, Belgium
Catherine Nicolis, Royal Institute of Meteorology of Belgium, Brussels, Belgium
Lode Wyns, Ph.D., Free University, Brussels, Belgium
Vasileios Basios, Université Libre de Bruxelles-ULB, Belgium
Frank Dubois, Université Libre de Bruxelles, Brussels, Belgium
Gregoire Nicolis, Free University, Brussels, Belgium
Madeleine Reis-Kautt, Universite Rene Descartes, Paris, France
Sevil Weinkauf, Department of Chemistry, TU, München, Germany
Luigi Carotenuto, MARS Center, Naples, Italy
Dario Castagnolo, MARS Center, Napoli, Italy
Chiara Piccolo, MARS Center, Napoli, Italy
Alexander Chernov, Marshall Space Flight Center, Huntsville, AL, United States

Developer(s)
European Space Agency (ESA), Noordwijk, Netherlands
University of Brussels (ULB-MRC and VUB), Brussels, Belgium
Pedeo Techniek, Oudenaarde, Belgium
Verhaert Design and Development, Antwerp, Belgium

Sponsoring Space Agency
European Space Agency (ESA)

Sponsoring Organization
Information Pending

Research Benefits
Information Pending

ISS Expedition Duration
October 2003 - October 2004

Expeditions Assigned
8,9

Previous Missions
ISS Expedition 6 was the first time the PromISS investigation had been performed in microgravity. PromISS-2 was sent to ISS during the ESA Cervantes mission in October 2003. The same setup was also used during a thirty day investigation in February 2004 for PromISS-3. PromISS-4 was operated during Increment 12.

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Experiment Description

Research Overview

  • PromISS-3 characterized the time crystallization experiments will take to reach equilibrium in a microgravity environment using the counter diffusion crystallization method.


  • PromISS-3 produces protein crystals for ground-based X-ray diffraction studies.


  • The holographic microscope will capture via video and measure several parameters of the growing protein crystals.

Description
One of the best-identified crystallization methods for proteins on earth and in space is the contra-diffusion technique that has been invented and developed by the team of Professor Garcia-Ruiz of the Granada University. With this technique, the protein solutions are placed in capillaries and the crystallization occurs thanks to a precipitating solution that is progressively diffusing along the capillaries. As the capillaries are small, the monitoring requests microscopic visualization that leads to a very limited depth of focus. It results that the direct observation of crystals in capillaries can give rise unfocused images. In order to overcome this classical limitation of optical microscopy, the Micro Gravity Research Center of the University of Brussels (B) developed a digital holographic microscope that allows reconstructing in depth by numerical means the objects under test. This instrument takes benefit of partial coherent illumination to eliminate the classical sources of noise inherent to the use a laser source and provides an image quality that is comparable to the one obtained with the best optical microscopes. As the 3D refocusing is made by numerical means, the technique is very interesting for automated experiment in space because it eliminates the need to focus on line the image of each crystal and it also compresses in a very important way the data to be stored. As the method is a true holographic one, it also records the optical phase information of the observed field. Therefore, this method gives an accurate measurement of the refractive index changes in the solution surrounding the crystals. For protein application, this is a very important point as the refractive index changes around crystal are a direct measure of the depletion zone that is expected to have a deep impact on the crystallization quality.

The major objective of the present experiment is to produce a detailed analysis and a quantitative interpretation of the relationship between the quality of the obtained crystals and the environment in which they are produced by the method of digital holography. The experiment aims to investigate the protein growth processes in weightless conditions using the counter diffusion technique, in order:

  • To measure the parameters of the growing protein crystals


  • To measure the composition changes (depletion zone) of liquid around the growing protein crystals.
Capillary counterdiffusion techniques have been developed for space experiments, and have proven to be very efficient for the crystallisation of proteins in general. To fully exploit the strengths of this technique, the crystallization processes in the capillaries should be fully characterized. The major objective of the present experiment is to produce a detailed analysis and a quantitative interpretation of the relationship between the quality of the obtained crystals and the environment in which they are produced by the method of digital holography.

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Applications

Space Applications
Information Pending

Earth Applications
Information Pending

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Operations

Operational Requirements and Protocols
The crew will unstow the PromISS hardware and perform the setup of the hardware inside the MSG. The sample wheel will be attached to the PromISS hardware. The PromISS session will be activated and the MSG video recorders will capture the video taken by the PromISS microscope. The crew will change the MSG video tapes daily, for seven days. The tapes and experimental cells are stowed for return to Earth. Following completion of the PromISS session, the hardware will be deactivated and stowed.

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Decadal Survey Recommendations

Information Pending

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Results/More Information

Of the 18 reactors flown for PromISS investigations on Expeditions 6, 7 and 8, twelve reactors produced crystals. This intermediate data from PromISS experiments are have shown that nucleation induction times are much longer in counterdiffusion experiments than in hanging drop experiments. There is non-negligible crystal movement during crystallization experiments on the ISS, a comparison of results between ISS and Foton should provide more information. The depletion zone model cannot explain the microgravity effect for all proteins and counterdiffusion techniques affect the crystal form. Crystal quality studies show gel can provide similar benefits as microgravity on the ISS, although mosaicities are higher in gel, and variation of quality of crystals is larger.

The use of counterdiffusion in some samples reduced nucleation rates significantly for proteins. This made it difficult to grow crystals in the timeframe. The scientists used supersaturation in an attempt to speed up the growth, which remedied the problem. On the other hand, some of the crystals showed improved diffraction properties and fewer impurities using the techniques of counterdiffusion.

Some of the reactors contained capillaries for the crystals to grow in. This was done to visualize the events in a capillary diffusion scenario. Crystals in the capillaries moved slowly, at speeds of one tenth of a millimeter per hour. They were reported to have moved up and down the capillaries, with some even moving perpendicular to them. Called the microgravity effect, the reason for this motion is not yet known. Most of the capillary crystals remained stationary.

The protein reactors containing Triose phosphate isomerase from Thermotoga maritima (TIM) are an example of proteins that grew in the designated timeframe. The crystals were easily removed from the capillary walls, showing that it was strong enough to resist movement but still very weak in general. In other reactors there were no capillaries for the crystals to grow on. These are believed to have been affected by the ISS environment, forming convective moments. PromISS four hopes to extend this library of model systems, as well as further explore the microgravity effect and other interesting results of the first phases of PromISS investigations.

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Results Publications

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Ground Based Results Publications

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ISS Patents

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Related Publications

    Dubois F, Requena MN, Minetti C, Monnom O, Istasse E.  Partial spatial coherence effects in digital holographic microscopy with a laser source. Applied Optics. 2004 February 10; 43(5): 1131-1139. DOI: 10.1364/AO.43.001131. PMID: 15008493.

    Dubois F, Minetti C, Monnom O, Yourassowsky C, Legros J, Kischel P.  Pattern recognition with a digital holographic microscope working in partially coherent illumination. Applied Optics. 2002 July 10; 41(20): 4108-4119. DOI: 10.1364/AO.41.004108. PMID: 12141510.

    Zegers I, Carotenuto L, Evrard C, Garcia-Ruiz JM, De Gieter P, Gonzales-Ramires L, Istasse E, Legros J, Martial J, Minetti C, Otalora Munoz F, Queeckers P, Cedric S, Van de Weerdt C, Willaert R, Wyns L, Yourassowsky C, Dubois F.  Counterdiffusion protein crystallisation in microgravity and its observation with PromISS (Protein Microscope for the International Space Station). Microgravity Science and Technology. 2006; 18-3/4: 165-169.

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Related Websites

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Imagery