1.1. Beamline overview

The VMXi beamline replaces the tunable MX beamline I02 (Allan et al., 2015; Grimes et al., 2018) which ended operations in August 2016. The original beamline layout has been extended by moving the sample position downstream and adding an additional experimental hutch and sample storage area. This configuration has provided a completely new sample area and allowed I02 to continue user operations during the build and installation of the VMXi beamline. The optical path has been reconfigured by repurposing components from the original I02 layout while extending specifications by adding a multilayer monochromator, a secondary source point and a pair of microfocus bimorph mirrors. In addition, the beamline is equipped with a new state-of-the-art detector and custom goniometry allowing very fast and precise data collections.

1.2. Operational overview

The VMXi beamline introduces a fundamentally different approach to how scientists use and interact with a beamline. The extensive level of automation introduced means that users no longer operate the beamline directly and have no specific beam time slot allocated. Instead, crystallization plates are stored at the beamline and users evaluate the crystallization results via a bespoke GUI in SynchWeb (Fisher et al., 2015). Data collection requests are detailed and submitted from the web interface and all requested beamline operations are carried out in a fully automated fashion. Data are processed automatically and the users are only notified when new data are available for review. This operating principle ensures optimal use of the available X-rays by decoupling the decision making of which data to collect and how to collect the data from beam time. It is matched to the unpredictable nature of the crystallization process where beam access is required once crystals appear rather than the other way around.

2.1. Monochromators

The beamline has a unique configuration with two separate monochromators which can be used depending on the experimental requirements: the original double-crystal monochromator (DCM) for narrow band-pass beam with lower flux and the new double-multilayer monochromator (DMM) for broader band-pass beam with higher flux. The monochromators are designed to enable beamline operation in either DCM or DMM configuration.

The DCM is an in-house upgrade of an original FMB Oxford design (Allan et al., 2015). The cryo-cooled Si(111) fixed-exit monochromator delivers a full energy range from 5 to 25 keV with a band-pass (ΔE/E) of <10⁻⁴ giving a flux at 13 keV of >2 × 10¹² photons s⁻¹ at the sample position. The DCM is the preferred standard option for MX data collection typically used for collecting oscillation data on single crystals and for measuring X-ray fluorescence for phasing purposes or low-dose X-ray scanning for locating small crystals.

The DMM is an in-house design described elsewhere (manuscript in preparation). This monochromator has two separate silicon crystals with two distinct multilayer coatings stripes each. The multilayers coatings are built of alternate layers of ruthenium (Ru) and boron carbide (B₄C) with layer thicknesses of 2.0 nm and 2.4 nm for the two stripes, respectively (Rigaku). By using either of the stripes, the DMM delivers beam across the energy range 10–25 keV with a band-pass (ΔE/E) of <5 × 10⁻² giving a flux at 13 keV of >10¹⁴ photons s⁻¹ at the sample position. The DMM is primarily used when a higher flux and broader band-pass is advantageous such as for raster scanning across large regions of a crystallization drop. The higher flux increases the speed at which large area scans can be completed, while the broader band-pass also improves the likelihood of correct cell parameters being assigned from still images by generating an effect akin to a small oscillation (Nave, 2014). Initial tests (see the Results section) suggest that, despite severe radiation damage, single-crystal oscillation data collections using the DMM can be processed with standard software and produce good quality data. We expect that, as the crystal lifetime is better defined and optimized, the DMM will also be routinely used for oscillation data collection.

We have also installed a low band-pass diamond filter upstream from the DMM to prevent low-energy radiation from the storage ring being reflected off the multilayer surfaces due to the very shallow operating angles used. This could cause low-energy contamination of diffraction as well as significant radiation damage to hardware further along the beam path. The specification and operational ranges for these diamond filters have been summarized in Table 1.

2.2. Focusing optics

2.2.2. Microfocusing mirrors

The beamline is equipped with a horizontal microfocus mirror (HMFM) and a vertical microfocus mirror (VMFM). The microfocus mirrors are located at the front of the new VMXi mini-hutch, 2 m and 1 m from the sample, respectively. To achieve optimal focused beam given the very tight location restrictions, both mirrors share a single vacuum vessel. The microfocus mirrors are super-polished SiO₂ substrate with 16 side-mounted piezo elements for shape adjustment (Alcock et al., 2015). The mirrors are polished to an elliptical shape to enable optimal focusing and defocusing and operate at 3 mrad. The mirrors have three different 12 mm-wide reflective areas to provide optimal reflectivity and harmonic rejection across the full energy range: one uncoated area used below 10 keV, a rhodium-coated area used between 10 and 18 keV, and finally a platinum-coated area providing optimal reflectivity at energies above 18 keV. Beam size and focal point can be modified by adjusting the voltage supplied to the 16 piezo actuators from the high-voltage power supply (CAEN ELS SRL). To achieve the focal range of 0 to 300 mm downstream from the sample and focal sizes ranging from 5 µm × 5 µm to 30 µm × 30 µm, the mirror voltages are operated between −1500 V and +1500 V (Fig. 2).

Figure 2 Current beam profile measured on a PIN diode while scanning a 10 µm-diameter aperture across the beam vertically and horizontally at the sample position. The insert shows an image of the attenuated beam on a YAG scintillator. The image depicts a 30 µm × 30 µm area and the beam is approximately 10 µm across. Further optimization of the beam focus is ongoing.

The mirror vessel also includes a `dissector' located upstream of the mirrors. The dissector has dual functionality and is designed to help with mirror optimization and also to act as a beam monitoring aperture. The small tungsten apertures of 10 and 20 µm in the horizontal and vertical planes are used to optimize the beam focus, using pencil-beam scans required for piezo voltage optimization (Sutter et al., 2012). A third aperture in each dissector (3 mm in the horizontal and 1.2 mm in the vertical) is used to trim the incoming beam to match the optical aperture of the mirrors. In addition, these beam-trimming apertures allow drain current measurement providing beam position feedback for the upstream optics.

2.3. Beam conditioning

As the beam converges towards the sample position a final set of elements are used to condition and fine-tune the beam. The beam passes through a set of slits, which are set to the nominal beam size to eliminate any unwanted scatter produced upstream. These slits can also be used to create a smaller aperture to reduce beam divergence. This will generate sharper Bragg reflections at the detector to improve the signal-to-noise ratio and resolve Bragg reflections for larger unit cells.

The beam can be attenuated by using aluminium and silver foils of various thickness (from 10 µm to 1.5 mm Al and 0.1 mm to 0.8 mm Ag). The filters are mounted on three motorized wheels each holding sets of ten foils [Fig. 3(a)] that can be placed in the beam path giving rise to 1000 individual filter combinations, giving a fine range of beam attenuation across the energy range 10–25 keV for both DCM and DMM beams. The beam position is carefully monitored using a four-quadrant diamond-based beam-position monitor (XBPM) (CIVIDEC Instrumentation GmbH) [Fig. 3(b)]. Attenuator filters and the XBPM are housed in a helium-filled environment to prevent oxidation and to aid motor cooling. The oxygen level in the chamber is monitored by using an InPro oxygen sensor (Mettler Toledo).

Figure 3 (a) The attenuator wheel assembly. The foils are held inside each of the inserts and the beam passes through the point where all three wheels intersect. (b) CIVIDEC XBPM mount. The four active pads used for monitoring beam intensity and position are coloured pink. (c) Sample shutter showing the stepped arrangement of the blades that allow for altering patterns of opening and closing. Blue is 50/50 and pink is 75/25. (d) Goniometer assembly showing the air-bearing, the scissor-like x–y translation stages, the z motion, the mirror interferometers and the tilt mechanism.

Having passed through the XBPM, the beam reaches the rotary shutter. The shutter is a rotating disk positioned perpendicular to the beam axis and is driven by an EC-type motor (Maxon Motors) [Fig. 3(c)]. The design of the shutter allows it to be used in a stop–start mode as a very fast conventional shutter with an opening time from fully closed to fully opened of only 50 µs. It is also possible to operate the shutter in continuous mode where the disk rotates and the beam is `chopped' eight times per revolution of the wheel. The motor is able to reach a speed of 5625 rev min<sup>−1</sup> enabling 750 Hz data collection frequency, matching the frame rate capabilities of the beamline detector. The blades on the chopper disk have a tiered design that allows the ratio of open/close time to be varied between 50/50 and re-focusing 70/25 in the current configuration by translating the chopper across the beam. To minimize background, the `chopper' mode can be synchronized with the detector to allow a sample to only be exposed to X-rays for a fraction of each collected frame rather than in a continuous exposure. This mode will be used when collecting serial crystallography data.

The final element before the sample is a set of three platinum–iridium apertures, 10, 50 or 200 µm in diameter (Agar Scientific) [Fig. 4(a)]. The apertures remove any unwanted scatter, helping to reduce background on the detector.

Figure 4 (a) Global view of the sample environment while a sample well is being imaged. (b) Overview of the VMXi experimental hutch, the Rock Imager storage units and the six-axis robot. (c) View of the 293 K sample local storage section. Empty and loaded sample holders are shown as well as a loaded sample holder in transit to the goniometer. The internal drum of the rotation load lock that allows samples to be loaded and unloaded through the closed radiation enclosure is also shown.

The beamstop is the final component in the beam path after the sample position before the detector [Fig. 4(a)]. In addition to preventing damage to the detector, the beamstop is also crucial for maximizing the quality of the diffraction data. The design reduces unwanted background while still allowing the collection of good quality data at low resolution. The current beamstop is a set of interlinked tubes as first described by Meents et al. (2017). In the VMXi configuration, the beamstop is made of three interlocked platinum tubes (400, 600 and 950 µm outer diameter) with the large tube plugged with gold wire and lead. It can be positioned 15 mm behind the sample, and at this distance the beamstop allows low-resolution reflections of approximately 93, 71 and 37 Å at energies of 10, 13 and 25 keV, respectively, to be recorded.

2.4. Detector

At the end of the beam path is an EigerX 4M detector (Dectris) (Johnson et al., 2012; Casanas et al., 2016; Tinti et al., 2017) [Table 2 and Fig. 4(a)]. This detector has an active area of 155.2 mm × 162.5 mm (W × H) with a pixel size of 75 µm × 75 µm. It is able to collect data continuously at 500 Hz and in short bursts of up to 30 s at 750 Hz. When operating at full flux the limiting factor is the photon count rate of 2.4 MHz per pixel as well as the bit depth of 12 bits. The detector performance will improve when VMXi starts operation with a new Eiger2X 4M detector at the beginning of 2019. This detector will have a significantly improved photon count rate, counter bit depth and effectively have no readout time. Further improvements will await the availability of a suitable charge integrating detector, which promises a tenfold increase in photon count rate.

2.5. Beam monitoring and feedback

For VMXi, all aspects of beam delivery and diagnosis are automated and three elements along the beam path provide feedback to maintain stable beam at the sample position. Firstly, drain current on the primary slits (located upstream from the monochromators) is used to monitor changes to the incoming beam from the storage ring. Secondly, changes in drain current on the dissector blades before the microfocus mirrors are used to adjust the pitch of the pre-focusing HFM. Finally, an X-ray beam-position monitor (CIVIDEC) upstream from the sample shutter is used to ensure that the beam is stable at the sample position by adjusting the horizontal and vertical pitch of the microfocus HMFM and VMFM. Monitoring currently runs at 10 Hz and feedback at 0.2 Hz but is disabled during data collection.

3.1. Sample storage

User plates are stored on the beamline in one of two modified Rock Imager 1000 units (Formulatrix, USA) operating at 277 K and 293 K, respectively [Fig. 4(b)]. These units store and automatically image up to 750 crystallization plates each. Additional features have been added to the VMXi Rock Imagers in collaboration with Formulatrix to allow full beamline integration.

The imaging modules in the Rock Imagers have been inverted to image crystallization plates through the bottom of the plates (Fig. 5). This means that crystallization drops are imaged from the same direction on and off the beamline enabling a more robust sample alignment. Furthermore, the Rock Imagers have been fitted with an automation load port (ALP) at the back of the units used for automatic transfer of plates between storage and the beamline for diffraction experiments [Fig. 4(b)].

3.2. Sample transfer

Sample transfer and data collection have been implemented, both at the software and hardware level, as separate concurrent processes so that sample loading and unloading does not affect the beamline output and vice versa. To achieve this, a radiation-safe transfer system (load lock) shown in Figs. 4(b) and 4(c) allows samples to be loaded in an out through the radiation enclosure while data are being collected. Furthermore, sample storage located inside the hutch allows twelve plates to be kept next to the goniometer (six at 277 K and six at 293 K), thus allowing quick exchange to the goniometer to maximize data collection time [Fig. 4(c)].

A sample transfer starts with the crystallization plate being delivered to the ALP in the Rock Imager. Once ejected via the ALP, the plate is picked up by a robot arm (RV-2FL MELFA, Mitsubishi Electric) mounted on a track running perpendicular to the beam path and alongside the Rock Imager storage units [Fig. 4(b)]. The robot arm delivers the plate to the load lock.

Once inside the shielding, the plate is picked up by a gantry robot (Sysmac Automation Platform, Omron) and transferred to the local storage area. Holding plates in adjustable high-precision plate holders [Figs. 3(d), 4(a) and 4(c)] ensures repeatable positioning on the goniometer improving accuracy when collecting data. Using individual holders reduces the time required to swap between data collection at 277 K and 293 K as it prevents waiting for a common holder on the goniometer to change temperature.

The waiting plate and holder are then transferred onto the goniometer [Figs. 3(d) and 4(a)]. This procedure allows an exchange time of plates of less than 20 s. To maintain the temperature of the 277 K plates and holders during data collection, the gantry robot mounts an additional cold cover over the plate, while in cold storage, before transferring the holder to the goniometer. This cold cover maintains the temperature of a plate at 277 K for up to 15 min on the goniometer.

All steps in the transfer are monitored to capture any anomalies and prevent damage to the plates or holders. Gripper sensors, barcode scanner and a proximity laser have been integrated to give the system the ability to detect and identify the status of transfer at every step of the process.

3.3. Goniometry

The crucial challenge for the goniometer design has been to achieve speed and precision to enable rapid translocation to the desired drop position within the SBS format while aligning and rotating micrometre-sized crystals in the beam (Table 2). The goniometer design enables data collection while oscillating the sample (omega scans) and data collection while moving the sample through the beam (area scans), and combinations of the two. The novel goniometer design [Fig. 3(d)] has a rotation axis (RT300L, Nelson Air) at the base aligned to the focal point of the beam. On this air bearing, we have placed the sample translation axes where a scissor-like design with two horizontal translations at the base (EC-type, Maxon Motors) actuate on two arms creating virtual horizontal and vertical sample axes as seen in Figs. 3(d) and 4(a). This motor configuration allows for very fast motions up to 100 mm s⁻¹ without bulky translation stages. Given the distance from the motor encoders to the sample and the dynamic nature of the motions we are using interferometers (Attocube) mounted at the base of the sample holder to account for any parasitic moves during data collection and make on-the-fly adjustments to correct them [Fig. 3(d)].

3.4. Sample imaging

To improve the success of matching the images marked by the users to those collected at the beamline, thus ensuring a good hit rate, the beamline is equipped with a Mag.x 125 vision system (Qioptiq) and a 6 MP Manta G609-B monochrome camera (Allied Vision) [Fig. 4(a)]. This system allows high-resolution images (>1 µm resolution) of entire drops (2 mm by 2 mm field of view) to be captured as shown in Fig. 5(a). The system also includes a second Optem FUSION lens (Qioptiq) with a 0.6 mm hole drilled through the centre, allowing the X-ray beam to pass through the vision system. This second lens is used for beam commissioning (Fig. 2). The lenses, which are co-axial to the beam, can be brought into position independently, and are replaced by a helium-filled tube to reduce air scatter when X-ray data are being collected (Fig. 4). Samples are illuminated using a LED RGB backlight (PHLOX).

5.1. General process

As described previously, the beamline operates automatically from data input parameters defined by the users. Users set up their crystallization experiments and review progress. Then, they mark regions or points to which data collection parameters can be assigned. Once these parameters are set up and the plate data collection has been requested, there is no further user involvement in the diffraction experiment until it comes to reviewing results and data processing.

The beamline is charged with loading and unloading the correct samples, locating the points or regions once the samples are on the beamline, preparing the beamline with the correct experimental settings, collecting the datasets and presenting the processing results.

5.2. User input via SynchWeb

An overview of the user interface in SynchWeb can be seen in Fig. 5. The interface currently captures all the required sample information for safe beamline operation prior to the crystallization plate arriving at the beamline. The information gathered also includes barcode references and storage temperatures that permit the beamline staff to load the samples in their correct location. Once sample barcodes are scanned in the storage units, the barcode links the plate to the stored information in the database. The crystallization plate is then imaged automatically according to a predefined schedule and users are notified via email whenever new imaging has been completed.

Figure 5 (a) The GUI tools in SynchWeb that allow users to review crystallization results, annotate drops, mark points and select regions for data collection. (b) SynchWeb view for setting data collection parameters and requesting collection of data.

As the users review their samples, they can mark points or regions on the sample images. These markers act as collection identifiers [Figs. 5(a) and 6(a)]. Once the user has marked one or more points or regions, diffraction data collection can be set up. The SynchWeb interface allows the user to assign a set of experimental data collection parameters to each point or region [Fig. 5(b)]. Once the users have supplied the required parameters the plate can be submitted for data collection. At this point, the plate joins a cohort of samples ready to be processed by the beamline for data collection.

Figure 6 Examples of data collection outputs in SynchWeb. (a, b) Images before and after data collection. Note the extreme radiation damage observed in the areas marked for data collection. (c, d) Area scan over groups of crystals. The overlaid heat-maps show where the highest number of diffraction spots were recorded. Similar overlay for (e) an area with crystalline material and long needles and (f) precipitated protein obscuring a crystal.

When the beamline is ready for a new plate to be transferred to a free sample holder, the sample loading routine queries the database for the next plate to load. A selection filter is employed to determine the next plate to be selected. The selection criteria can be optimized to improve data output and to make best use of beam time available. Once a plate has been selected, it is conveyed to the beamline for data collection.

5.3. Sample alignment

The alignment of a drop for data collection is a fully automated multi-step process. The first step is to position the drop by scanning it through the focal plane and look for in focus features. The second step is to create an extended-focus image by merging twelve images taken every 20 µm as the drop is moved through the focal plane. The third step is to align this new image and the original image to determine any displacement and thereby convert marked points and regions to the goniometer position.

For oscillation data collection we include an additional analysis for in focus features in a narrow region around the marked point to ensure that the marked point is on the rotation axis and stays in the beam during oscillation. Finally, we do also have to shift the drop position before starting data collection to correct for the optical distortion caused by the plastic and liquid. This is a plate-specific parameter and varies between 30 and 180 µm for the plates tested on the beamline so far.

5.4. Data processing and outputs via SynchWeb

When the requested diffraction data collections for a plate have been completed and are available for review and processing, the user is notified by email. At the moment, automatic data processing relies on existing pipelines used by other MX beamlines at DLS. For example, Xia2 (Winter et al., 2013) image analysis is used to analyse grid scans and produce heat maps that can be displayed in SynchWeb [Figs. 6(c)–6(f)]. Xia2 is also used to index, integrate and scale the individual oscillation datasets using DIALS (Winter et al., 2018) (Table 3).

Table 3 Data collection statistics using the DMM beam (five thaumatin crystals from the same crystallization drop) Data collections wedges of 60° collected in 1 s each. All datasets were trimmed to only use the best wedge of data and resolution was cut to obtain complete data in all resolution shells. Data reprocessing of each wedge was undertaken using the available tools in SynchWeb. Merging of the final dataset was achieved using DIALS. Dataset number in SynchWeb [see Fig. 5(a)] Dataset 1 Dataset 12 Dataset 24 Dataset 30 Dataset 35 All (2.2 Å cut-off) All (2.0 Å cut-off) Images collected 1–600 1–600 1–600 1–600 1–600 30000 30000 Images used for processing 100–600 1–450 200–600 1–250 200–400 18000 18000 Space group P41212 P41212 P41212 P41212 P41212 P41212 P41212 Unit-cell parameters (Å) a = b = 58.7, c = 151.6 a = b = 58.57, c = 151.28 a = b = 58.54, c = 151.25 a = b = 58.54, c = 151.28 a = b = 58.62, c = 151.62 a = b = 58.6, c = 151.4 a = b = 58.6, c = 151.39 Resolution range (Å) 46.42–1.87 (1.92–1.87) 54.62–1.86 (1.91–1.86) 38.20–1.86 (1.91–1.86) 28.74–1.87 (1.92–1.87) 50.54–2.15 (2.21–2.15) 54.65–2.2 (2.26–2.2) 54.65–2.2 (2.26–2.2) Rmerge (all I+ and I−) 0.055 (0.139) 0.068 (0.173) 0.082 (0.131) 0.074 (0.369) 0.095 (0.462) 0.089 (0.170) 0.095 (0.312) Rmeas (all I+ and I−) 0.061 (0.197) 0.080 (0.173) 0.097 (0.186) 0.092 (0.522) 0.122 (0.653) 0.093 (0.187) 0.095 (0.376) Rpim (all I+ and I−) 0.026 (0.139) 0.041 (0.173) 0.026 (0.131) 0.054 (0.369) 0.068 (0.453) 0.025 (0.074) 0.026 (0.204) Total No. of reflections 44178 (97) 47524 (89) 35031 (55) 21362 (55) 16433 (447) 152069 (4513) 163393 (1901) No. of unique reflections 12188 (84) 14435 (71) 11821 (50) 11294 (45) 8114 (396) 13793 (848) 16951 (750) Mean I/σ(I) 17.2 (3.1) 11.9 (1.9) 7.3 (2.2) 8.1 (2.8) 4.8 (1.1) 20.5 (9.6) 17.9 (4.6) Mn(I) half-set correlation CC(1/2) 0.997 (0.956) 0.993 (0.768) 0.990 (0.967) 0.986 (0.873) 0.994 (0.811) 0.993 (0.974) 0.993 (0.846) Completeness (%) 53.9 (5.3) 62.5 (4.2) 52.0 (3.2) 49.8 (2.8) 54.5 (38.6) 97.4 (83.6) 97.4 (57.2) Multiplicity 3.6 (1.2) 3.3 (1.3) 3.0 (1.1) 1.9 (1.2) 2.0 (1.1) 11.0 (5.3) 9.6 (2.5) Mean (χ2) 0.91 (0.00) 0.97 (0.66) 0.94 (0.00) 0.85 (0.00) 0.97 (1.01) 0.94 (0.82) 0.93 (0.75)

Due to the high volume of data and the complexity of the decisions needed to arrive at a final dataset, further developments in software are ongoing. They will eventually allow users to easily review hundreds of partial datasets across multiple plates. The outcome can either feed back into the crystallization process or piece together all the partial information to a complete dataset and structure determination.