Acoustic Focusing of Protein Crystals for In-Line Monitoring and Up-Concentration during Serial Crystallography

Area of Science:

• Structural biology
• Biophysics
• Materials science

Background:

• Serial femtosecond crystallography (SFX) is crucial for high-resolution protein structure determination using X-ray free-electron lasers (XFELs).
• Reliable sample delivery of microcrystals remains a bottleneck, often causing nozzle clogging and limiting data collection.

Purpose of the Study:

• To develop an improved sample delivery method for SFX using acoustic focusing.
• To enable real-time online characterization of microcrystal size and shape.
• To enhance microcrystal concentration for increased data collection efficiency.

Main Methods:

• Developed acoustic 2D focusing of protein microcrystals within capillaries using a piezoelectric actuator.
• Created a standing wave perpendicular to crystal flow to focus microcrystals into a single line.
• Utilized a high-speed camera for real-time imaging and characterization.
• Designed a splitting union for microcrystal up-concentration.

Main Results:

• Successfully focused lysozyme microcrystals into a single line within a silica capillary.
• Characterized acoustic contrast factor, focus size, and coaxial flow lines.
• Achieved microcrystal up-concentration by a factor of at least five, with potential for up to 200-fold concentration.
• Demonstrated real-time online characterization of crystal properties.

Conclusions:

• Acoustic 2D focusing offers a robust solution for microcrystal delivery challenges in SFX.
• The developed system enhances sample delivery reliability and data collection rates at XFELs.
• This technology has potential applications for serial crystallography at synchrotrons and for proteins available in limited quantities.