Microcrystallography on a Chip*

Figure 3: P11 Dewar plunger for automatic chip preparation.

Figure 4: Automatic chip scanning software at P11.

(in collaboration with Diamond Light Source, Paul Scherrer Institut and University of Oxford)

Motivation

At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques such as nylon loops do not fulfill the requirements of these new experiments. They typically hold only one crystal, significantly contribute to background scattering, and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 μm and smaller (see Fig. 1).

Figure 1: (a, b) Microcrystals of Operophtera brumata CPV18 polyhedrin Si-microchip. (c, d) Polyhedrin structure determined from chip-based microcrystallography at 100 and 1.5 Å resolution. Data were collected at the microfocus beamline I24, Diamond Light Source, UK (Image taken from Ref. 1).

Chip Design

The chip is entirely made from single crystalline silicon and consists of a frame with dimensions of 4 × 2.5 mm2 and a thickness of 130 μm and an inner membrane part of 1.5 × 1.5 mm2 with a thickness of 10–30 μm, depending on the design (see Fig. 2). The membrane part features a few hundred to several thousands of micropores. In general the size and pitch of the pores can be matched to the crystal and X-ray beam size, respectively. Pore sizes down to 1 μm, with a pitch of 3 μm, have already been successfully realized by us. The micropores are generated by electron beam lithography and subsequent reactive ion etching. The chip is designed to work both at cryogenic and ambient temperatures. The chip dimensions are chosen such that the chip is compatible with most conventional open-flow cryocoolers installed at macromolecular crystallography beamlines. In order to further prevent conductive heating of the chip by its mount adapter, which is typically at room temperature, the chip is glued onto the tip of a thermally insulating plastic pin attached to a standard mount used in macromolecular crystallography. Using this approach and with chip dimensions smaller than the inner cold gas stream of the cryojet no ice forms on the chip or on the base. The chip design further allows easy mounting onto the goniometer since conventional cryotools such as vials and cryotongues can be used. It can be mounted at any crystallography beamline and is compatible with most sample changing robots.

Figure 2: Design of the Si-microchip (Image taken from Ref. 1).

Sample Loading

A suspension of the microcrystals, with a volume of typically 1–3 μL and a crystal density of 1000–2000 microcrystals per microliter, is pipetted onto the chip. By attaching a wedge of filter paper from the lower side of the chip, the mother liquor is soaked off through the micropores. Microcrystals larger than the pore diameter are retained and stay on the upper side of the chip. During sample loading, the chip is constantly exposed to an air stream with controlled humidity to prevent the crystals from drying out and thereby losing their diffraction properties. In case of more viscous microcrystal suspensions, sample loading and liquid removal can be supported by air suction. Directly after crystal loading, the chip is flash-frozen by plunging it into liquid nitrogen. Due to the good thermal conductivity of silicon and the fact that most of the mother liquor is removed, we expect that crystals smaller than 20 μm do not require any cryoprotection and can be directly flash-frozen. High occupation densities of the pores with crystals can be achieved using this procedure. At P11, an inhouse-designed plunge-freezing device for automatic sample preparation including removal of the excessive mother liquor and subsequent chip-freezing is provided to our users (see Fig. 3).

Data Collection

The resulting periodic arrangement of the crystals in the pores allows for an automated raster scan approach for X-ray data collection. Appropriate software is available at P11 beamline control and enables fully automated data collection conducting both translational and rotational movements of the chip (see Fig. 4). Owing to a highly brilliant microfocused beam with a photon flux of 1.3 × 10^13 ph/s at 12 keV and a spot size of 4 × 9 µm² (v × h) or 2 × 1011 ph/s with a spot size of 1 × 1 µm² (v × h) X-ray diffraction data from crystals with sizes in the range as small as a few hundred nanometers up to several tens of micrometers can be obtained with fully automated measurements. By efficiently removing the surrounding mother liquor during sample loading the signal-to-noise ratio is furthermore improved, which is especially important in the case of nanocrystals due to their typically weak diffraction signals. Since it is made of single crystalline silicon the chip itself practically does not contribute to the background signal.

* Images and plain text are partially taken from Ref. 1. This work is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0).

References
1) A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering,
P. Roedig et al., Sci. Rep. 5, 10451 (2015).

2) Room-temperature macromolecular crystallography using a micro-patterned silicon chip with minimal background scattering,
P. Roedig et al., J. Appl. Cryst. 49, DOI: 10.1107/S1600576716006348 (2016).

For further information please contact: