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

Serial femtosecond crystallography (SFX) has become one
of the
standard techniques at X-ray free-electron lasers (XFELs) to obtain
high-resolution structural information from microcrystals of proteins.
Nevertheless, reliable sample delivery is still often limiting data
collection, as microcrystals can clog both field- and flow-focusing
nozzles despite in-line filters. In this study, we developed acoustic
2D focusing of protein microcrystals in capillaries that enables real-time
online characterization of crystal size and shape in the sample delivery
line after the in-line filter. We used a piezoelectric actuator to
create a standing wave perpendicular to the crystal flow, which focused
lysozyme microcrystals into a single line inside a silica capillary
so that they can be imaged using a high-speed camera. We characterized
the acoustic contrast factor, focus size, and the coaxial flow lines
and developed a splitting union that enables up-concentration to at
least a factor of five. The focus size, flow rates, and geometry may
enable an upper limit of up-concentration as high as 200 fold. The
novel feedback and concentration control could be implemented for
serial crystallography at synchrotrons with minor modifications. It
will also aid the development of improved sample delivery systems
that will increase SFX data collection rates at XFELs, with potential
applications to many proteins that can only be purified and crystallized
in small amounts.