Small-angle X-ray scattering (SAXS) allows to analyze the nanometer-sized internal structure of a sample. However, X-ray sources with traditional collimation systems are only capable to deliver beams which are typically millimeter-sized. This range of X-ray spot sizes prevents the precise analysis of small or heterogeneous samples due to the signal averaging over the illuminated area.
This situation improved tremendously with the greatly increased peak brilliance of state-of-the-art synchrotrons and with the advent of specialized techniques for the generation of microfocused X-rays. 4,16 Due to the pioneering work at dedicated focused X-ray beamlines, a variety of focusing principles has been developed that involve components like Kirkpatrick-Baez (KB) crossed mirrors or -multilayers, capillaries, Fresnel optics, wave guides or compound refractive lenses (CRL) and together with advances in high-precision positioning systems, these techniques now enable X-ray foci down to the nanometer range. 11,16-26 Consequently, samples can now be analyzed with a much higher spatial resolution and greater precision. This capability allows to distinguish between differently structured domains in a material of which the scattering signal previously had been averaged.
However, the focus spot size is not the only thing to consider for experiments with focused X-ray beams. It is also very important to consider the divergence of the beam as well as the overall photon flux. 20 The relation between these three parameters can be imagined as a triangle because all of them are interrelated. For example, it is possible to generate very small-sized X-ray beams using CRL-focusing optics. This also increases the photon flux at the focus position tremendously. However, this is only possible at the cost of a higher beam divergence and consequently a loss of resolution. It is also possible to have a small beam with low divergence for an increased resolution, but this might only be possible at the cost of photon flux which results in longer measurement times. If photon flux and divergence are both important, the minimum X-ray spot size has to be larger with a decrease of spatial resolution, although the beam size would still be in the low micrometer range. An example overview over the spot sizes and its influence on divergence and flux for different focusing techniques is shown in Fig. 1.
Figure 1 Comparison of different X-ray microfocusing techniques and their spot size-dependent influence on flux and divergence. The focusing types include capillaries (top), Kirkpatrick-Baez (KB) crossed mirrors (middle) and compound refractive lenses (CRL, bottom). (Figure from 20, Copyright IOP Publishing)
Today’s dedicated microfocus X-ray beamlines at 3rd generation synchrotrons, like for example P03 (PETRA III at DESY, Hamburg, Germany) or ID13 (ESRF, Grenoble, France), also take the focusing distance from the optics to the sample into account. This leads to a lower divergence at a given focus spot size while a high photon flux is maintained. In the end, it is a question of the optimum combination of focusing settings for a given sample or experimental setup. In case of a microfluidic X-ray experiment, the maximum divergence of the microfocused beam is dictated by the channel height which typically ranges between 50 µm and 100 µm. In most of our microfluidic SAXS experiments, the X-ray beam size was adjusted to spot sizes around 10 µm by 10 µm to guarantee a low divergence and maintain a high photon flux.
An example which clearly demonstrates the benefits of the high spatial resolution of microfocused X-ray beams is the analysis of thin cellulose- or high-performance polymer fibers27-29 A fiber’s structure can be mapped in great detail which also provides information about the fibers internal structure. Another very interesting example for a biological sample system are spider silk fibers. 22,30-34 These in vitro and in vivo studies revealed how the spider silk fiber’s nanostructure changes during the spinning process, during its elongation or under the influence of and how this affects the fiber’s tensile strength and micro-structural properties.
The smaller X-ray beam size is also important for grazing-incidence small-angle scattering (GISAXS) because the microfocus-illuminated area is much smaller compared to conventional X-ray beams, enabling better spatial scanning resolution. 35
Another example where the use of a microfocused X-ray beam greatly improves the spatial resolution is SAXS microtomography. 36-40 This measurement technique reveals the internal three-dimensional structure of a sample, like for example a high performance polymer fiber, by rotating it during the detailed mapping with the X-ray beam. Due to the Nyquist-Shannon sampling theorem and the microfocused X-rays that enable the mapping of much smaller volumes, the sample scans require much less images (and therefore shorter scanning times) for a given resolution compared to tomographic scans with larger beams. 41,42
The ongoing development of X-ray sources leads to increasingly brilliant and intense beams.
4 This can lead to new problems concerning the sample: the maximum X-ray dose before the sample degrades. Biological samples or soft matter are just two examples of sample systems which are susceptible to radiation damage. 43 A protein crystallographic case study demonstrated that it is possible to increase the sample’s resilience to beam damages through freezing. 44 Obviously, this cryogenic approach is impractical for liquid samples or solutions that freeze below the targeted temperature, but it is a very useful method for protein crystals.
However, this study also revealed that the successful collection of a crystallographic data set is only possible up to a certain X-ray dose because otherwise the sample degrades before the data set is obtained. 44 This maximum dose dictates a minimum crystal size in the micrometer
range which is very limiting for the protein-structure determination at traditional X-ray sources like synchrotrons or lab sources. The required minimal crystal size could be reduced by the use of microfocused X-rays in combination with motorized sample handling that enable precise crystal scans, but it is still an extremely challenging task to grow ’large’ micrometer-sized protein single crystals in the first place. 43,45
One possible approach to avoid the beam damage for liquid samples or solutions is to use a continuous sample stream minimizing the sample residence time in the volume of the X-ray focus. This approach requires a sample environment that allows to control liquids with very high precision and reproducibility on the micrometer scale. If this was not the case, the possibly high sample consumption rates of macroscopic flow systems would make the required sample amounts impractical. This is where the combination of microfocused X-ray beams and X-ray compatible microfluidic devices becomes important and demonstrates its strengths. 9,46-48 The challenges and benefits of X-ray compatible microfluidic sample environments as well as first examples of experiments will be discussed in further detail in the following chapter.
Another approach for overcoming the maximum dose limit of solid and liquid samples becomes available with the advent of X-ray free electron lasers (XFEL). 49-51 This next generation of X-ray sources solves the radiation damage problem by generating highly intense femtosecond X-ray pulses and applying the principle of ‘diffraction before destruction’. 6,52 Each generated X-ray pulse is so intense that a full diffraction pattern is collected with this single shot while its pulse length is so short (femtosecond range) that the diffraction pattern is generated before the sample is destroyed by Coulomb explosion. Consequently, the successful collection of diffraction patterns requires a fresh sample with each single pulse. However, the realization of an efficient and reliable way of generating a continuously replenishing sample stream is a very challenging task. 53,54 This is where the microfluidic liquid jet devices, which are introduced in more detailed in chapter 7.3, offer great potential as a sample environment for XFELs and other pulsed laser experiments due to the flexible design control and fast fabrication routines of these devices.