Crystallography on a chip - without the chip: sheet-on-sheet sandwich

Abstract

Crystallography chips are fixed-target supports consisting of a film (for example Kapton) or wafer (for example silicon) that is processed using semiconductor-microfabrication techniques to yield an array of wells or through-holes in which single microcrystals can be lodged for raster-scan probing. Although relatively expensive to fabricate, chips offer an efficient means of high-throughput sample presentation for serial diffraction data collection at synchrotron or X-ray free-electron laser (XFEL) sources. Truly efficient loading of a chip (one microcrystal per well and no wastage during loading) is nonetheless challenging. The wells or holes must match the microcrystal size of interest, requiring that a large stock of chips be maintained. Raster scanning requires special mechanical drives to step the chip rapidly and with micrometre precision from well to well. Here, a `chip-less' adaptation is described that essentially eliminates the challenges of loading and precision scanning, albeit with increased, yet still relatively frugal, sample usage. The device consists simply of two sheets of Mylar with the crystal solution sandwiched between them. This sheet-on-sheet (SOS) sandwich structure has been employed for serial femtosecond crystallography data collection with micrometre-sized crystals at an XFEL. The approach is also well suited to time-resolved pump-probe experiments, in particular for long time delays. The SOS sandwich enables measurements under XFEL beam conditions that would damage conventional chips, as documented here. The SOS sheets hermetically seal the sample, avoiding desiccation of the sample provided that the X-ray beam does not puncture the sheets. This is the case with a synchrotron beam but not with an XFEL beam. In the latter case, desiccation, setting radially outwards from each punched hole, sets lower limits on the speed and line spacing of the raster scan. It is shown that these constraints are easily accommodated.

Keywords: Mylar sandwich chip; XFEL; fixed target; high throughput; low dose; room-temperature data collection; serial crystallography.

Figure 1

(a) Loading the SOS sandwich based on the chip holder (Owen et al., 2017 ▸); (b) loaded SOS sandwich. A larger than necessary 25 µl sample volume is shown being loaded in these images.

Figure 2

A silicon chip exposed to an unattenuated XFEL beam at 7.3 keV photon energy at SACLA. The beam (1.4 × 1.6 µm FWHM) was centred in the wells (7 × 7 µm). Nevertheless, significant damage to the chip was observed. The chip is shown from the back after exposure. The scattered silicon powder resulted in significant diffraction. In some chips, the accumulated stress was so large that the chip fractured. The orange scale bar corresponds to 100 µm.

Figure 3

(a) Typical diffraction image for lysozyme microcrystals, 2–3 µm in size, mounted in the SOS sandwich. The corner of the detector corresponds to 2.0 Å resolution, with strong diffraction peaks (b). The diffuse scattering ring (4.7 Å) originates from the two 2.5 µm thick Mylar sheets. XFEL beam at 7.3 keV photon energy, 480–500 µJ pulse energy, 70% beamline transmission, 10 fs duration, (1.4 × 1.6 µm FWHM) XFEL spot, 30 Hz pulse repetition rate.

Figure 4

(a) Microscope image of the SOS sandwich loaded with haemoglobin microcrystals (brown granular material) after exposure to unattenuated XFEL pulses at 7.3 keV photon energy. (b, c) Enlarged views focused on the crystal layer (b) and on the edges of through-hole burned by the XFEL (c). Here, the stepwise raster scan was 125 µm on centres horizontally and 250 µm vertically. The orange scale bar corresponds to 100 µm in (a) and 20 µm in (b) and (c).

Figure 5

(a) Background of the silicon chip and SOS sandwich. The signal was calculated as the median of 1000 diffraction images (hits) of haemoglobin microcrystals mounted in the respective device for SFX data collection. The plot of the radial distribution is shown in (b).