New Device Will See Details of Protein Crystal Growth

STS-86 to carry apparatus to Mir on first flight

[Methods][Apparatus][Operations]

Jan. 5, 1998 update: High-resolution JPGs added for print media.

A new apparatus for measuring details of how protein molecules move through a fluid and then form crystals will be carried to space station Mir by the STS-86 Space Shuttle mission, set for launch in September 1997. This will be the first flight for the Interferometer Protein Crystal Growth (IPCG) apparatus.

Space Shuttle missions since 1985 have proven the improved value of protein crystals grown in space. However, some details of how crystals grow - and what conditions lead to the best crystals - remain a mystery. IPCG will investigate this area.

The experiment is managed by the MSFC Microgravity Research Program Office, Biotechnology Project Office. The program manager is Ron Porter. The principal investigator is Dr. Alexander McPherson of the University of California at Riverside. Co-investigators are William Witherow and Dr. Marc Pusey of MSFC.

All PCG investigations ultimately depend on the movement of large molecules (macromolecules) through a fluid as the concentration of protein solution is raised until the protein molecules contact each other and form a crystal much like stacking bricks in an orderly manner. PCG experiments have shown that growth also depends on the temperature, salt (precipitant) concentration, and acid/base balance (pH). Further, it may also be controlled by the molecules grouping together, in aggregates such as octamers (eight molecules), without forming crystals. Impurities also control the growth rate.

PCG studies on Earth are often thwarted by convective flow since the attachment of molecules to the crystal depletes the concentration. This depleted volume is less dense and tends to rise, thus causing the solution to flow across the crystal surface. The low-g environment of space eliminates convective flows caused by differences in density.

Differences in density also affect how the fluid bends light (by changing the index of refraction). Thus, an optical system which shows variations in density can be a powerful diagnostic technique for distinguising how temperature, concentration, or pH differences drive crystal growth.

[Interferometer][Fluid handling system][Flight data system]

The IPCG comprises three major systems - interferometer, six fluid assemblies, and flight data - designed to produce images showing density changes in a fluid as a crystal forms.

The interferometer employs a Michelson-Morley phase-shift interferometer (derived from a design by A.A. Michelson and Edward Morley to measure the speed of light in 1887). Light is made of waves, and waves that are out of step with each other will form light-and-dark interference patterns. A cube beamsplitter takes a beam of light, splits it and passes half through a medium and the other half through the same distance in open space, the recombines the two beams. Mirrors and other optical devices split, return, and recombine the light beam. Passing through the medium slows that part of the light beam, so the recombined beam is out of phase and produces an interference pattern that carries information about the disturbance.

In the IPCG, the glass crystal growth cell, only 1 mm thick, is silvered on the back to act as the mirror on the experiment leg of the light path. Light from a deep red diode laser (670 nm) is divided by a cubical beam splitter, goes to the experiment cell and a reference mirror and then back to the beamsplitter where they travel as two beams, one polarized vertical and one polarized horizontal.

The new beam is enlarged by 20-power, long-working-distance microscope which directs it into a phase shifter. This is a liquid crystal cell that can retard slightly the horizontal polarized beam without affecting the vertical polarized beam. Retardation occurs along the plane of an electric voltage applied across the liquid crystals. A Dove prism folds the light through a 180 deg. turn (extending the light path while keeping the design compact) and delivers it through a polarizing filter to a video camera. The polarizing filter is oriented at 45 deg. (i.e., evenly tilted to both beams), so the two beams produce an interference pattern. The computer, via the waveform generator, controls the phase modulator to produce varying interference patterns.

Each fluid handling system is a self-contained plastic assembly enclosing two pairs of 4 mL supply syringes, a waste receptacle, a test cell, and associated plumbing, plus mechanisms to inject the fluids and to position the test cell. The syringe pairs each consist of a solution syringe (dissolved lysozyme) and a precipitant syringe (salt water to prompt crystal growth). Each pair feeds through valves to mix in a T-intersection and then flow into the experiment cell. From there, the solution goes into a waste receptacle. The entire system is filled before launch to prevent air bubbles that could disrupt the experiments. The system contains enough fluid for three experiment runs, including flushing the test cell between each run. The crew operates the fluid system by a hand crank which depresses the syringe plungers. The test cells are mounted on a tilt platform that allows the crew to reposition the cell in three dimensions and tilt in two dimensions to produce a clear image.

The flight data system includes a 486-based laptop computer and is powered by the Mir Payload Unit Panel. The computer has a 23 cm (9-inch) display panel and a 1.3 gigabyte hard drive. It is equipped with an electronic waveform generator, to control the phase shifter, and a video frame grabber, to capture images during the experiments. A 26 cm (10.4-inch), 480x640 pixel, color flat-panel display allows the crew to view the video from interferometer. The video system uses the glovebox's data and video interface drawer. In addition, an ATR-4 solid-state data recorder will record ambient temperatures in the glovebox during experiments. Data may be stored up to a year before readout.

IPCG will be carried aboard the Shuttle in a middeck locker, then transferred to Mir after docking. The glovebox provides a small, enclosed work space in which flight crews handle fluids or materials which should not be released into the cabin. The glovebox includes ports so the crew can insert experiment hardware, and utilities such as electrical power and video.

To conduct IPCG experiments, the crew will install the IPCG optical system and fluid system in the Mir Glovebox, and connect the system to the laptop computer (outside the glovebox).

Each IPCG experiment set involves injecting the first half of syringe pair No. 1, waiting for nucleation, injecting the first half of No. 2 (1st experiment run), injecting the rest of No. 2 (2nd run), then injecting the rest of No. 1 (3rd run), and replacing the fluid system with the next system to be used. At the start of each run, the crewman selects a crystal, adjusts the focus and adjusts the tilt platform to minimize fringes, then lets the system automatically collect images and data. Six fluid sets will be carried, allowing a total of 18 experiment runs. The 12 syringe pairs contain different combinations of protein and precipitant so that a wide variety of growth conditions may be studied.

Each experiment will last from 4 to 72 hours, depending on the protein concentration. Because the objective is to study growth, all crystals go into the waste syringe after use. Results will come from approximately 4,050 video frames captured and stored in the computer until it is returned to Earth by STS-89 in early 1997. From these images, investigators will be able to determine which factors which have the greatest influence on crystal growth, and what must be controlled to produce the best crystals.