Sample-minimizing co-flow cell for time-resolved pump-probe X-ray solution scattering.

A fundamental problem in biological sciences is understanding how macromolecular machines work and how the structural changes of a molecule are connected to its function. Time-resolved techniques are vital in this regard and essential for understanding the structural dynamics of biomolecules. Time-resolved small- and wide-angle X-ray solution scattering has the capability to provide a multitude of information about the kinetics and global structural changes of molecules under their physiological conditions. However, standard protocols for such time-resolved measurements often require significant amounts of sample, which frequently render time-resolved measurements impossible. A cytometry-type sheath co-flow cell, developed at the BioCARS 14-ID beamline at the Advanced Photon Source, USA, allows time-resolved pump-probe X-ray solution scattering measurements to be conducted with sample consumption reduced by more than ten times compared with standard sample cells and protocols. The comparative capabilities of the standard and co-flow experimental setups were demonstrated by studying time-resolved signals in photoactive yellow protein.

Lipidico Injection Protocol for Serial Crystallography Measurements at the Australian Synchrotron.

A facility for performing serial crystallography measurements has been developed at the Australian synchrotron. This facility incorporates a purpose built high viscous injector, Lipidico, as part of the macromolecular crystallography (MX2) beamline to measure large numbers of small crystals at room temperature. The goal of this technique is to enable crystals to be grown/transferred to glass syringes to be used directly in the injector for serial crystallography data collection. The advantages of this injector include the ability to respond rapidly to changes in the flow rate without interruption of the stream. Several limitations for this high viscosity injector (HVI) exist which include a restriction on the allowed sample viscosities to >10 Pa.s. Stream stability can also potentially be an issue depending on the specific properties of the sample. A detailed protocol for how to set up samples and operate the injector for serial crystallography measurements at the Australian synchrotron is presented here. The method demonstrates preparation of the sample, including the transfer of lysozyme crystals into a high viscous media (silicone grease), and the operation of the injector for data collection at MX2.

Repeatability and accuracy of a foot muscle strength dynamometer.

Toe flexor strength is a pivotal biomechanical contributor for effecting balance and gait. However, there are limited reports that evaluate measurement accuracy and repeatability of this important attribute. Dynamometers are designed to measure force which can be used to derive joint torque if the perpendicular distance to the joint axis is known. However, an accurate and reliable measurement method to assess the ability of the toe flexor muscles to produce torque, is lacking. Here we describe a new device and method, designed to quantify the toe flexor torque developed at the metatarsal phalangeal joint. We evaluate measurement bias and the ability of the instrument to consistently measure what it is supposed to measure (Interclass Correlation Coefficient). Results suggest that our device is an accurate tool for measuring angle and torque with a small (0.10° and 0.07 Nm, respectively) bias. When tested for reliability and repeatability in measuring toe flexor torque (n = 10), our device showed high interclass correlation (ICC = 0.99), small bias (-1.13 Nm) and small repeatability coefficient (CR = 3.9). We suggest mean bias and CR to be reported for future measurement methods and our protocol used as standard approach to measure maximal toe flexor torque.

Improved radiation dose efficiency in solution SAXS using a sheath flow sample environment.

Radiation damage is a major limitation to synchrotron small-angle X-ray scattering analysis of biomacromolecules. Flowing the sample during exposure helps to reduce the problem, but its effectiveness in the laminar-flow regime is limited by slow flow velocity at the walls of sample cells. To overcome this limitation, the coflow method was developed, where the sample flows through the centre of its cell surrounded by a flow of matched buffer. The method permits an order-of-magnitude increase of X-ray incident flux before sample damage, improves measurement statistics and maintains low sample concentration limits. The method also efficiently handles sample volumes of a few microlitres, can increase sample throughput, is intrinsically resistant to capillary fouling by sample and is suited to static samples and size-exclusion chromatography applications. The method unlocks further potential of third-generation synchrotron beamlines to facilitate new and challenging applications in solution scattering.