Suspending sample droplets with sound waves

TinyLev offers a cheap and portable way to use acoustic levitation at synchrotron beamlines

The blue levitating beads represent samples in this prototype, developed by Dr Pete Docker at Diamond Light Source

Acoustic levitation suspends matter using acoustic radiation pressure to balance the force of gravity. It has potential applications in crystallography, spectroscopy, chemistry, and the study of organisms in microgravity. However, conventional acoustic levitation systems rely on Langevin horns, which are large and expensive pieces of equipment that are complicated to set up. TinyLev, initially developed by researchers at the University of Bristol, is a small single-axis non-resonant acoustic levitator constructed from off-the-shelf components. In work recently published in Scientific Reportsengineers at Diamond led by Dr Pete Docker used the TinyLev system to dispense and contain sample droplets in protein crystallography experiments. Their novel method facilitates efficient X-ray data acquisition in dynamic studies at room temperature.

Sample delivery for Macromolecular Crystallography

Macromolecular Crystallography (MX) is a powerful technique for assessing protein structures. Samples are routinely cryogenically cooled to minimise the radiation damage from powerful X-ray beams, but this can induce structural artifacts and locks the normally flexible protein into static condition. New techniques that allow effective data acquisition at room temperature and allow for reaction dynamics to be probed would, therefore, provide a valuable tool for structural biologists.

At X-ray Free Electron Lasers (XFELs), the brilliance of the XFEL pulse allows the collection of a single diffraction image to be recorded before the protein crystal is destroyed, removing the need for the sample to be cryo-cooled. This had led to the development of sample delivery systems that can feed large quantities of protein crystals into the beam, at room temperature - techniques that are now being exploited at synchrotrons

Experiments at both synchrotron and XFEL sources have shown that acoustic levitation can effectively present MX samples to the beam at room temperature, and is not destructive to the sample. Acoustic levitation also avoids presenting any non-sample material to the beam, as there is no requirement for external supports. However, conventional acoustic levitation systems rely on equipment that is expensive and complex and requires a temperature- and humidity-controlled environment. These large systems also impart considerable energy to the sample, causing it to heat up.

Proteins in a spin at Diamond

Left: Photograph showing the TinyLev system mounted on the I24 beamline with the X-ray beam path marked with a yellow dashed arrow. Components as labelled: (A) High-magnification viewing system, (B) X-ray scatter-guard, (C) levitating drop, (D) beamstop (out of position), (E) TinyLev Transducer array, (F) backlight (retracted during data collection), (G) sample positioning stage. Right: Model of the acoustic levitation system (E) used in this work annotated with key dimensions and showing the focal point of the transducer array.
Left: Photograph showing the TinyLev system mounted on the I24 beamline with the X-ray beam path marked with a yellow dashed arrow. Components as labelled: (A) High-magnification viewing system, (B) X-ray scatter-guard, (C) levitating drop, (D) beamstop (out of position), (E) TinyLev Transducer array, (F) backlight (retracted during data collection), (G) sample positioning stage. Right: Model of the acoustic levitation system (E) used in this work annotated with key dimensions and showing the focal point of the transducer array.

Researchers at the University of Bristol developed the TinyLev system to make acoustic levitation cheaper and simpler so that this useful technique is more widely available.

Elizabeth Dye is a joint PhD student with Diamond and Nottingham Trent University. Together with her Diamond Supervisor Peter Docker, Elizabeth has developed a TinyLev system for presenting MX samples to Diamond's room temperature beamlines. It is built from off-the-shelf components - including motion detectors designed for home security systems - and only cost around £100. The portable device is extremely low-power, running at 1-2 W, and imparts no heat to the sample.

Growing protein crystals often requires the use of additives with high surface tension. This can cause problems in droplet delivery systems, with the crystal solution remaining attached to the pipette tip rather than dropping into the levitation system. In these experiments, the engineers overcame the issue by giving the droplets a coating of oil, which had the added benefit of significantly reducing evaporation rates.

This acoustic levitation system holds steady in the X-ray beam, but also imparts enough spin to allow the collection a complete, high-quality, rotation style dataset from a single crystal. It will enable researchers to efficiently and routinely carry out MX experiments.

Pete Docker says:

Working with co-supervisor Danny Axford on the I24 beamline, we've developed a cheap, simple and portable acoustic levitation sample delivery system. The potential benefits to researchers are enormous. We're thrilled to have already been able to solve protein structures with this novel method, and have recorded the entries in the protein database.

The future of acoustic levitation

The small size and weight of the TinyLev system mean it is easily positioned on beamlines using the existing beamline sample positioning stages. The team has plans to install a second device on VMXi and believe the system is suitable for deployment on other high-intensity X-ray sources operating in ambient conditions.

The present version collects complete datasets in as little as 0.7s. Their goal is to reduce this to 0.1s, a data collection rate that allows some biological processes (such as ligands bonding to proteins) to be seen, and which would enable researchers to expand time-resolved studies at Diamond, reducing the need to visit an XFEL.

They want to develop a better understanding of the physics involved in acoustic levitation, to make the samples rotate faster and to make the drops smaller. The surface area to mass ratio is critical for acoustic levitation, and theoretical estimates suggest the minimum droplet size is in the picolitre range.

There is also the potential for future work to explore automated delivery of droplets, which would significantly increase throughput, and to investigate more effective control of the droplet motion to optimise diffraction data acquisition.

 

To find out more about the project, please contact Dr Pete Docker: peter.docker@diamond.ac.uk

Related publication:

Morris RH et al. Non-contact Universal Sample presentation for Room temperature Macromolecular crystallography Using Acoustic Levitation. Scientific Reports 9, 12431 (2019). DOI:10.1038/s41598-019-48612-4.