Multi-channelin situdynamic light scattering instrumentation enhancing biological small-angle X-ray scattering experiments at the PETRA III beamline P12

Abstract

Small-angle X-ray scattering (SAXS) analysis of biomolecules is increasingly common with a constantly high demand for comprehensive and efficient sample quality control prior to SAXS experiments. As monodisperse sample suspensions are desirable for SAXS experiments, latest dynamic light scattering (DLS) techniques are most suited to obtain non-invasive and rapid information about the particle size distribution of molecules in solution. A multi-receiver four-channel DLS system was designed and adapted at the BioSAXS endstation of the EMBL beamline P12 at PETRA III (DESY, Hamburg, Germany). The system allows the collection of DLS data within round-shaped sample capillaries used at beamline P12. Data obtained provide information about the hydrodynamic radius of biological particles in solution and dispersity of the solution. DLS data can be collected directly prior to and during an X-ray exposure. To match the short X-ray exposure times of around 1 s for 20 exposures at P12, the DLS data collection periods that have been used up to now of 20 s or commonly more were substantially reduced, using a novel multi-channel approach collecting DLS data sets in the SAXS sample capillary at four different neighbouring sample volume positions in parallel. The setup allows online scoring of sample solutions applied for SAXS experiments, supports SAXS data evaluation and for example indicates local inhomogeneities in a sample solution in a time-efficient manner. Biological macromolecules with different molecular weights were applied to test the system and obtain information about the performance. All measured hydrodynamic radii are in good agreement with DLS results obtained by employing a standard cuvette instrument. Moreover, applying the new multi-channel DLS setup, a reliable radius determination of sample solutions in flow, at flow rates normally used for size-exclusion chromatography-SAXS experiments, and at higher flow rates, was verified as well. This study also shows and confirms that the newly designed sample compartment with attached DLS instrumentation does not disturb SAXS measurements.

Pulsed Electron-Electron Double Resonance (PELDOR) and Electron Spin Echo Envelope Modulation (ESEEM) Spectroscopy in Bioanalysis

“…a monodisperse solution (Jeffries et al 2016). Today, individual light scattering techniques and setups allow to manipulate the solution composition during the experiment (Meyer et al 2012), investigate sample solutions in flow, to investigate a sample suspension at different neighboring positions in parallel (Falke et al 2018), determine the net surface charge of molecules (Ware 1974) or even allow to obtain limited shape and structure information in situ (Cantor and Schimmel 1980;Minton 2007;Haghighi et al 2013;Schubert et al 2015) as outlined in following sections of this chapter. Especially for in situ DLS instruments with freely adjustable laser probe the alignment of the optics, i.e.…”

“…In contrast to cross-correlation DLS, the term multi-channel DLS was recently used for a setup that allows to record multiple DLS data sets in parallel (Falke et al 2018). The setup uses one laser and records the scattering data in different neighboring and statistically independent positions of a sample solution close to the focal point of the laser.…”

“…As discussed DLS is most suitable to verify sample homogeneity to match a high standard of sample quality in nano particle sample preparation towards imaging and manufacturing techniques. Consequently, a few setups and devices have been developed to continuously monitor and score the sample quality in situ and in parallel to other experimental procedures, including growth of macromolecular crystals (Meyer et al 2012), SAXS (Schwamberger et al 2015;Falke et al 2018), asymmetrical field flow fractionation (Sitar et al 2017) and to score nanocrystal suspensions to be measured at XFEL radiation sources in the future as well as in context of other experimental procedures in biochemistry, genetics, material science, sorting and chemical synthesis.…”

A multi-channel in situ light scattering instrument utilized for monitoring protein aggregation and liquid dense cluster formation

“…It is applicable to different sample environments with comparably low sample consumption. Particle size distributions can be investigated in real-time and optionally in flow mode [30, 31]. The intensity pattern of light scattered by particles in solution is correlated with itself within short time intervals to determine the autocorrelation function (ACF), which allows determining the diffusion constant of the particles via an inverse Laplace transform and allows calculating hydrodynamic radii (R h ) via the Stokes-Einstein equation [32].…”

“…Today's most common DLS applications include the characterization of suspensions in life sciences, as well as industrial quality control in material science and biotechnology. One example in structural biology is scoring and optimization of protein solutions prior to crystallization and SAXS experiments [31, 33]. For the experiments described, we designed, constructed and applied a novel and up to now unique in situ DLS instrument, which allows simultaneous recording of scattered light in up to eight different positions approx.…”