Small Angle X-ray Scattering (SAXS)

Small and Wide-Angle X-ray Scattering (SAXS and WAXS) are analytical techniques used to study the structure and interactions of materials in the 1 to ~300 nm range. Using USAXS (Ultra SAXS) this range can be extended to over a micron. With these closely related techniques, information can be gained on particle sizes and shapes, order an orientation, particle interactions and internal structures. Measurements can either be done on bulk materials, or on the surface only when using GISAXS or GIWAXS (Grazing Incidence SAXS or WAXS).

The materials typically studied include polymers, metals, colloids, liquid crystals and biological samples e.g. proteins and RNA/DNA (also referred to as Bio-SAXS). Bio-SAXS has recently gained significant interest in research as well as industry. It has proven to be a valuable technique for the investigation of flexible and disordered proteins and it is complementary to high resolution techniques such as  Protein Crystallography and Electron Microscopy.

SAXS measurements can be done on many different types of samples: gels, liquids, solids, etc. Furthermore, little or no sample preparation is required. These two characteristics enable measurements in the native state and in-situ measurements. This gives invaluable insight into the behavior of the analytes in real-life conditions.

In general, the scattered signal is recorded at diffraction angles below 6° and the sample is positioned at a long distance from the detector. This means that the scattered signal is often weak. Therefore, SAXS measurements benefit greatly from the use of a high brilliance X-ray source such as the Excillum MetalJet. Higher signal means that the weak scattering effects become stronger, more visible and thus more readily studied. Especially time dependent studies (such as the kinetics of particle formation), in-situ measurements (for example under tensile or shear stress) and measurements on weak scatterers like most proteins, benefit hugely from the MetalJet’s high brilliance. Moreover, the use of a high brilliant source reduces the measurement time hereby increasing the amount of measurements that can be done.

Application examples for Small Angle X-ray Scattering (SAXS)

In-lab SEC-SAXS for structual investigation of protein samples

Small-Angle X-ray Scattering (SAXS) is a prevalent technique in structural biology for the investigation of protein structure and interactions. It gives efficient access to parameters like the radius of gyration, molecular weight, degree of folding, a low-resolution model of the 3D shape of proteins, protein multimers and complexes.

Often, biological samples consist of a complex mixture of structurally different molecules and complexes in dynamic equilibria. SAXS measurements of such a mixture result in an average structure of the species in solution which can be very difficult – if not impossible – to deconvolute. An efficient solution to this problem is offered by the use of Size Exclusion Chromatography (SEC). By fractionating the mixture based on the size of the components, you can measure the SAXS signal of the individual species.

The combination of SEC with SAXS does however put a huge time constraint on the SAXS measurements. Short measurement times are required to prevent the separated samples from reaching a new equilibrium. Short measurements on weakly scattering samples could previously only be done at the synchrotron. With the introduction of the MetalJet source, this measurement capability is now also available in the laboratory as was demonstrated with the Xenocs BioXolver.

The images below present a case where Bovine Serum Albumin (BSA) was separated into the monomeric and dimeric form. The UV trace shows the separation into monomers and dimers. After separation, these species are measured with SAXS using an exposure time of only 60 seconds. As illustrated below, these short measurements provide sufficient statistics both for concentrations of 8 mg/ml as well as down to 1 mg/ml to establish the most important parameters and obtain a 3D structure.

Application note kindly provided by Application Scientist at Xenocs SAS using a BioXolver equipped with Metaljet X-ray source.

Bucciarelli, S. et al. Size-exclusion chromatography small-angle X-ray scattering (SEC-SAXS) of water soluble proteins on a laboratory instrument, J. Appl. Crystallogr. 2018


Fibers are an important component in many natural and man-made materials ranging from clothes to technologically advanced materials like optical fibers. Many natural fibers, such as for example spider silk, are extremely strong and durable. To mimic the properties of this type of materials in synthetic fibers, a thorough understanding of the fiber structure is required. Small-Angle X-ray Scattering (SAXS) provides an excellent means to obtain information on parameters such as porosity, crystallinity, and the size, shape and dispersity of structural units as well as their orientation. Together, these properties dictate the mechanical properties of the fibers. The technique is also well suited to monitor how these properties change during production and under load and stress.

Especially for the analysis of very thin fibers and for in situ measurements, a high intensity, low divergence X-ray source is advantageous. The high intensity of the MetalJet source allows short measurement times, even for weakly scattering fibers. For time resolved measurements, such as tensile stretching or the monitoring of an extrusion process, the high intensity is indispensable. The low divergence of the MetalJet ensures good separation between signals, which in turn gives better defined reflections and allows for more accurate integration of the intensities.

The result of a SAXS measurement on a fiber can be seen in the 2D scattering pattern of rat tail tendon above. These thin fibers are a common standard for the calibration of SAXS instruments. Rat tail tendons consist of collagen, which has a primary spacing of about 67 nm and produces a SAXS pattern with equidistant peaks. The above scattering patterns were acquired by application scientists at Bruker AXS using a Nanostar instrument, equipped with an three different X-ray sources: a sealed-tube, solid anode microfocus source, a rotating anode, and an Excillum MetalJet (200W at 70kV). These experiments show that the MetalJet gives an intensity gain of more than 50 times compared to sealed microfocus tubes.

Data courtesy of J. Lange, A. Schwamberger and K. Erlachen of Bruker-AXS.

Biological (Bio-SAXS): Structural characterization of an apoptosis-regulating protein

Recently, a MetalJet source mounted on a Bruker Nanostar was used to help identify the structure of the Bcl-xL protein. This member of the Bcl-2 family plays a role in the regulation of programmed cell death (apoptosis) by governing mitochondrial calcium ion transport. Previous research has shown that Bcl-xL exists in different topological states, mainly as dimers, but structural studies have so far been focused on the monomeric form. Furthermore, two different states of Bcl-xL were known, but the transition between these water-soluble and membrane-bound states remained elusive.

Oligomerization has been shown to be an important step in the process of membrane insertion for other Bcl-2 family members. Therefore, the researchers of Nanyang Technological University, A*STAR, University of Louisville, Rosalind Franklin University of Medicine and Science, and Kyung Hee University set out to resolve the structure of Bcl-xL dimers.

The Bcl-xL dimerization was induced by the addition of a mild detergent (n-Octyl β-D-Maltoside) and the resulting structure was studied by a combination of crystallography, nuclear magnetic resonance (NMR) and Small-Angle X-ray Scattering (SAXS). Protein crystallography showed that the detergent-treated Bcl-xL forms a dimer through three-dimensional domain swapping (3DDS) of helixes α6-α8 between two monomers. The formation of stabile dimers was confirmed by SAXS analysis as indicated by the lack of aggregation which can be seen in the Guinier plot (insert in figure a) and the maximum dimensions (shown in figure b). Furthermore, SAXS measurements yield a low-resolution solution structure of the dimer which fits well with the crystal structure.

The SAXS patterns were acquired for 1 mg/ml and 3mg/ml solutions using a liquid gallium-based MetalJet source with the sample-to-detector distance set to 670 mm using a total acquisition time of 30 minutes.

Metallic nanoparticles

Colloidal metal nanoparticles are a class of materials with myriad applications: drug delivery, medical imaging, catalysis and for antimicrobial purposes, to name just a few. They also show potential as strain gauges. A monolayer of colloidal gold nanoparticles on a flexible substrate offers an interesting alternative to currently used resistive strain gauges. Researchers at the Slovak Academy of Science and STU Centre for Nano-diagnostics show that they provide a fast and linear response is a wide strain range.

Small-Angle X-ray Scattering (SAXS) gives insight into the electrical response of this new type of gauges by efficiently probing the structural changes of the nanoparticle assemblies induced by stress on the gauge. The use of a high intensity MetalJet X-ray source uniquely enables the researchers to study this system out of equilibrium, during the stretching of the foil. The images below show the 2D scattering pattern (a) of the used gold particles suspended in hexane. Fitting of the radially averaged 1D curve (b) reveals the shape and diameter of the particles.

The MetalJet is available with a gallium-rich (l = 1.34 Å) and an indium-rich (l = 0.51 Å) target. These low wavelengths result in lower absorption, and thereby an increase in the scattering signal. This is especially interesting for high-Z materials such as most metals, which are challenging to measure using a copper source. Furthermore, using these short wavelengths, smaller structures can be probed, air scattering is minimized, which makes in-air measurements more attractive.

These properties, together with the high flux, ensure that short measurements of only 10 seconds, provide sufficient statistics for the analysis of the gold particle monolayers on Mylar foil exposed to uniaxial stress. The in-situ measurements allow the correlation of interparticle distance with the simultaneously measured stress-strain curve, hereby enabling comparison of the macroscopic and microscopic properties of the gauge.

The SAXS measurements were performed in vacuum on a Bruker-AXS Nanostar instrument equipped with a Ga liquid metal-jet X-ray microfocus source.


The investigation of polymers is one of the main areas of focus of the Soft Matter Analytical Laboratory (SMALL) at the University of Sheffield. A £2 million infrastructure investment enabled them to purchase a state-of-the-art SAXS instrument with a gallium-based MetalJet X-ray source in 2016. The first publication with their new equipment was a study on the reversible addition–fragmentation chain transfer (RAFT) copolymerization of styrene with N-phenylmaleimide, resulting in the formation of block copolymer nano-objects with a potential application as foam stabilizers.

The researchers at SMALL showed that polymerization-induced self-assembly (PISA) of this copolymer gives different morphologies depending on the ratio between the two polymer blocks. In their article, they present a novel synthesis method using a less toxic cosolvent compared to previous studies: methyl ethyl ketone versus the previously used 1,4-dioxane. Transmission electron microscopy (TEM) revealed vesicular morphologies, similar to what was observed in their previous studies with other PISA formulations. Using Small-Angle X-ray Scattering (SAXS) however, they were able to obtain structural information about the internal structure of these vesicles. This way, they found that in the current system they obtain oligolamellar vesicles, whereas the use of a different stabilizer block results in unilamellar platelet-like particles. Furthermore, SAXS confirms the morphology of the self-assembled copolymers (see figure above) and gives access to statistically relevant information about the size and shape of the self-assembled polymer chains.

The SAXS instrument, equipped with a liquid gallium MetalJet, was the first of its kind to be installed in the UK. The intensity of the X-rays is about a hundred times higher than what could be achieved with the old SAXS instrument. Dr. Oleksandr Mykhaylyk, the facility manager, says that this enables them to analyze their samples in several minutes, rather than the several hours it took with the old instrument.


Recent developments in Bio-SAXS
Advanced crystallography


MetalJet for X-ray analytical applications


SAXS publications

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