Research Article: Sample delivery for serial crystallography at free-electron lasers and synchrotrons

Date Published: February 01, 2019

Publisher: International Union of Crystallography

Author(s): Marie Luise Grünbein, Gabriela Nass Kovacs.


Current developments and challenges for serial sample delivery at synchrotrons and X-ray free-electron lasers are reviewed, including the new megahertz repetition-rate machines, with an emphasis on liquid injection and high-viscosity extrusion.

Partial Text

The unprecedented properties of X-ray free-electron lasers (XFELs) enable novel research in a variety of scientific fields (Pellegrini, 2016 ▸). An XFEL delivers as many photons in a single pulse (of femtosecond duration) as a third-generation synchrotron source does in an entire second. Moreover, the radiation is highly coherent (Pellegrini et al., 2016 ▸). For macromolecular crystallography, this 109-fold increase in peak brilliance means that high-resolution diffraction patterns can be collected from weakly diffracting objects such as tiny protein crystals (Colletier, Sawaya et al., 2016 ▸; Gati et al., 2017 ▸), including membrane proteins (Liu et al., 2013 ▸). Importantly, the ultrashort pulse duration allows a diffraction pattern to be collected before the onset of radiation damage, even at room temperature (Neutze et al., 2000 ▸; Chapman et al., 2014 ▸). Thus, essentially damage-free data can be obtained even for radiation-sensitive samples (Young et al., 2016 ▸; Suga et al., 2017 ▸). XFELs also facilitate time-resolved (TR) measurements, including studies of irreversible reactions (Aquila et al., 2012 ▸; Tenboer et al., 2014 ▸; Nogly et al., 2016 ▸; Nango et al., 2016 ▸). The short pulse duration delivers temporal resolution down to the sub-picosecond range (Barends et al., 2015 ▸; Pande et al., 2016 ▸; Coquelle et al., 2017 ▸). Also, the ability to use small crystals is crucial for both optically and chemically triggered reactions. Optical absorbance in a protein crystal is high, owing to the high protein concentration, and this limits optical penetration to at most a few micrometres (Barends et al., 2015 ▸). In a similar fashion, the diffusion of chemical triggers into such crystals is often a rate-limiting step, with smaller crystals giving proportionally shorter diffusion times (Schmidt, 2013 ▸).

Microcrystals suspended in their mother liquor or in a suitable carrier medium can be injected into the X-ray interaction region as a continuous jet or as droplets synchronized with the XFEL pulse. Different techniques for jet and droplet injection have been developed over the years, enabling the injection of samples of various viscosity ranges. Since these techniques operate in different flow regimes, they also present different opportunities and challenges (Weierstall, 2014 ▸) regarding sample efficiency, use at high repetition-rate XFELs and TR experiments.

Fixed-target techniques are another means of serially delivering fresh sample for each X-ray exposure. Here, the crystals are immobilized on a substrate, which is then scanned through the X-ray beam. An inherent advantage of this approach is that in principle the geometry and crystal distribution can be arranged such that every crystal on the substrate is probed. Each step in a raster scan must clearly move beyond the area affected by previous probe pulses, which is particularly important at an XFEL. Numerous solid-support approaches have been developed over the years and, depending on the design, they mainly vary in (i) the X-ray background, which can be caused by the substrate itself and by excess mother liquor, (ii) the extent to which they support high-throughput data collection in terms of maximal data-collection rate and high hit rate, (iii) whether only specific crystal sizes or shapes can be accommodated, (iv) crystal handling, i.e. crystal growth directly on the substrate/off the substrate, preventing crystal dehydration during loading or data collection, and (v) whether they can be used at room or cryogenic temperature.

Despite the increasing variety of sample-delivery techniques, there is no universal technique of choice for serial crystallo­graphic data collection at XFEL or synchrotron sources. Instead, the suitability of a specific technique strongly depends on the investigated system and the experimental aim and conditions. We expect serial sample delivery to remain a rapidly developing field as the number of next-generation synchrotrons and XFELs is increasing, as will the user community.