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. 2022 Sep 29;2(4):100081.
doi: 10.1016/j.bpr.2022.100081. eCollection 2022 Dec 14.

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

Mukul Sonker  1   2 , Diandra Doppler  1   2 , Ana Egatz-Gomez  1   2 , Sahba Zaare  2   3 , Mohammad T Rabbani  1   2 , Abhik Manna  1   2 , Jorvani Cruz Villarreal  1   2 , Garrett Nelson  3 , Gihan K Ketawala  1   2 , Konstantinos Karpos  2   3 , Roberto C Alvarez  2   3 , Reza Nazari  2   3 , Darren Thifault  1   2 , Rebecca Jernigan  1   2 , Dominik Oberthür  4 , Huijong Han  5 , Raymond Sierra  6 , Mark S Hunter  6 , Alexander Batyuk  6 , Christopher J Kupitz  6 , Robert E Sublett  6 , Frederic Poitevin  6 , Stella Lisova  6 , Valerio Mariani  6 , Alexandra Tolstikova  4 , Sebastien Boutet  6 , Marc Messerschmidt  1   2 , J Domingo Meza-Aguilar  1   2 , Raimund Fromme  1   2 , Jose M Martin-Garcia  7 , Sabine Botha  2   3 , Petra Fromme  1   2 , Thomas D Grant  8 , Richard A Kirian  2   3 , Alexandra Ros  1   2
Affiliations

Electrically stimulated droplet injector for reduced sample consumption in serial crystallography

Mukul Sonker et al. Biophys Rep (N Y). .

Abstract

With advances in X-ray free-electron lasers (XFELs), serial femtosecond crystallography (SFX) has enabled the static and dynamic structure determination for challenging proteins such as membrane protein complexes. In SFX with XFELs, the crystals are typically destroyed after interacting with a single XFEL pulse. Therefore, thousands of new crystals must be sequentially introduced into the X-ray beam to collect full data sets. Because of the serial nature of any SFX experiment, up to 99% of the sample delivered to the X-ray beam during its "off-time" between X-ray pulses is wasted due to the intrinsic pulsed nature of all current XFELs. To solve this major problem of large and often limiting sample consumption, we report on improvements of a revolutionary sample-saving method that is compatible with all current XFELs. We previously reported 3D-printed injection devices coupled with gas dynamic virtual nozzles (GDVNs) capable of generating samples containing droplets segmented by an immiscible oil phase for jetting crystal-laden droplets into the path of an XFEL. Here, we have further improved the device design by including metal electrodes inducing electrowetting effects for improved control over droplet generation frequency to stimulate the droplet release to matching the XFEL repetition rate by employing an electrical feedback mechanism. We report the improvements in this electrically triggered segmented flow approach for sample conservation in comparison with a continuous GDVN injection using the microcrystals of lysozyme and 3-deoxy-D-manno-octulosonate 8-phosphate synthase and report the segmented flow approach for sample injection applied at the Macromolecular Femtosecond Crystallography instrument at the Linear Coherent Light Source for the first time.

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Conflict of interest statement

A.E.G., J.C.V., and A.R. hold a patent on electrical droplet stimulation in a 3D-printed device.

Figures

Figure 1
Figure 1
Schematic of the droplet injection setup and injector assembly details. (A) Representation of the elements required for droplet injection. (B) Representation of the capillary coupled droplet injector device consisting of 1) the 3D-printed droplet generator, 2) electrodes, 3) optical fibers, 4) fiber-capillary aligner device, 5) He gas supply line, and 6) 3D-printed GDVN. (C) Photograph of a completely assembled device showing the same elements as the schematics in (B).
Figure 2
Figure 2
Droplet generator design and electrical stimulation (A) Schematic of the 3D-printed droplet generator showing the Y-intersection of flow channels (yellow) for oil and sample as well as electrodes (dark gray) for electrical stimulation. (B) Image snapshots of droplet generation at 120 Hz with KDO8PS crystal-laden buffer without electrical stimulation. (C) Image snapshots of droplet generation at 120 Hz with the same sample as in (B) but with electrical stimulation. Electrical pulses of 200 V amplitude and 2 ms duration were employed. The wetting effect of the buffer solution on the inner channel wall is apparent at 3.2 ms as indicated by the red dashed line. This effect causes the ability to adjust frequency and delay in reference to XFEL pulses. The dark speckles in the sample droplet in (B) and (C) are the KDO8PS crystals in solution.
Figure 3
Figure 3
Electrical feedback and data aquisition (A) Schematic of the experimental setup and instrumentation for the electric feedback mechanism deployed for continuous triggering at a given frequency and delay to the XFEL pulse. (B) Selected plots of real-time data stream recorded for XFEL reference (red), droplet detector (blue), electrical trigger (purple), and resultant droplet frequency (green) during the LV14 beamtime.
Figure 4
Figure 4
Representation of XFEL signal (red), droplet signal (blue) as recorded by the droplet detector, and electrical stimulus (green) (top) and resulting waterfall plots (bottom). (A) Frequency variation is apparent when droplets were generated based on flow-rate ratio differences between oil and aqueous phase alone without electrical stimulation. Top: the depicted time interval of ≈40 ms shows droplets generated at a frequency of 116 Hz, slightly lower than the required 120 Hz. Bottom: the waterfall plot shows frequency variation patterns over a larger 8 s period. (B and C) Stimulated droplet generation frequency with active feedback aiming for a programmed delay of (B) 2 ms and (C) 7 ms to the XFEL reference signal. Lysozyme injection buffer (L2) served as the sample in (A)–(C). The color scale represents the voltage signal recorded with the photodetector (in volts). The feedback mechanism “pins” the droplet leading-edge position at the desired delay to the XFEL reference in (B) and (C) successfully. Note that the droplet appears wrapped around the XFEL reference in the waterfall plot in (C), since the droplet leading edge is successfully programmed to 7 ms, but the droplet width is >2 ms.
Figure 5
Figure 5
Representative data traces showing continuous droplet generation with electrical feedback mechanism as achieved in the HERA chamber: (A) Representation of XFEL reference signal, applied voltage pulse (trigger), and droplet signal (top) and resultant waterfall plot (bottom) for droplets generated at 120 Hz. The sample generating aqueous-phase droplets contained lysozyme crystals. (B) Representation of XFEL reference, applied voltage pulse (trigger), and droplet signal (top), and resultant waterfall plot (bottom) for continuously triggered droplets at 120 Hz. The sample generating aqueous droplets contained KDO8PS crystals. The duration of the trigger events and their delay to the XFEL reference are depicted by white bars in the waterfall plots. The colors in (A) and (B) correspond to the droplet detector signal measured in volts. Note the different “shape” of the droplet trace in (B), such that the heat plot shows voltages up to 9 V.
Figure 6
Figure 6
Snapshot of the droplet and crystal hit rates showing an increase in both rates upon application of electrical stimulation for lysozyme crystal sample (L2) during LV14.
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