Femtosecond x-ray diffraction from an aerosolized beam of protein nanocrystals

TL;DR

This paper demonstrates near-atomic-resolution x-ray diffraction from aerosolized protein nanocrystals using an x-ray free-electron laser, reducing background noise and enabling studies of small crystals with high-speed capabilities.

Contribution

It introduces an aerosol injector method with helium carrier gas for high-resolution diffraction of protein nanocrystals, improving background reduction over traditional liquid nozzles.

Findings

Achieved near-atomic-resolution Bragg diffraction from aerosolized crystals.

Reduced x-ray scattering background by using helium instead of liquid.

Suitable for high-repetition-rate x-ray free-electron lasers.

Abstract

We demonstrate near-atomic-resolution Bragg diffraction from aerosolized single granulovirus crystals using an x-ray free-electron laser. The form of the aerosol injector is nearly identical to conventional liquid-microjet nozzles, but the x-ray-scattering background is reduced by several orders of magnitude by the use of helium carrier gas rather than liquid. This approach provides a route to study the weak diffuse or lattice-transform signal arising from small crystals. The high speed of the particles is particularly well suited to upcoming MHz-repetition-rate x-ray free-electron lasers.

Equations

$$D=\frac{N_p}{A\times R},$$

$$H_1=\exp (-\sigma D{R}_X)\sigma D{R}_X\approx \sigma D{R}_X$$

$$H_1^{\pm }\approx \frac{\pi }{4}D(d_X\pm d_p{)}^2{R}_X$$

Full text

{synopsis}

A new particle-injection approach is demonstrated that achieves very low background in the measurement of diffraction from macromolecular nanocrystals by using an aerosol-focusing injector with an x-ray free-electron laser.

1 Introduction

Serial femtosecond crystallography (SFX) allows the structural analysis of macromolecular crystals that may be too small or weakly scattering to study with synchrotron radiation sources. In order to record any measurable diffraction signal, such samples would require exposures far in excess of limits imposed by x-ray induced radiation damage when using conventional sources. With typical pulse energies of about 1 mJ, or $10^{12}$ photons, and durations of about 10 fs, pulses from x-ray free-electron lasers (XFELs) overcome this limit by producing diffraction data before the onset of most damage processes [Neutze:Nature406:752, Chapman:Nature470:73, Boutet:Science337:362]. Furthermore, XFELs enable novel time-resolved studies with femtosecond temporal resolution, angstrom spatial resolution, all at physiological temperatures. A variety of prominent results from SFX measurements are summarized in recent reviews and special issues [Spence:RPP75:102601, Schlichting:COSB22:613, Barty:ARPC64:415, Patterson:CR20:242, Schlichting:IUCRJ2:246, Johansson:TBS42:749].

The large increase in x-ray fluence afforded by the ability to outrun damage not only increases the diffraction signal from the sample, but it also increases the diffuse scattering from the medium transporting the crystal to the beam. Many SFX measurements were, therefore, performed on microcrystals that were large enough and ordered well-enough to produce intense Bragg reflections that could be measured in the presence of the diffuse background. Such examples helped the rapid adoption of the technique. The use of such crystals, usually with volumes greater than $\unit{1}{\micro\metre^{3}}$, enabled a broad range of sample delivery methods to be used depending on the nature of the experiment, such as liquid microjets [DePonte:JPD41:195505, Weierstall:PTRSB369:20130337], viscous extrusion injectors [Weierstall:NatComm5:3309] or solid substrates [Frank:IUCRJ1:95, Roedig:Nm14:805]. This flexibility is in stark contrast to efforts to record high-resolution coherent diffraction patterns from non-crystalline samples [Seibert:Nature470:78, Kuepper:PRL112:083002, Aquila:SD2:041701, Yang:PRL117:153002]. Without the amplfication of the diffraction signal due to periodicity, objects such as molecules, viruses, and other particles produce only weak scattering signals. Non-crystalline samples must, therefore, be delivered to the x-ray focus in a vacuum environment and in isolation from other potential scattering sources. This can be achieved, for example, through aerodynamic focusing of aerosolised particles [Bogan:NanoLett8:310, Bogan:AST44:i, Bogan:JPB43:194013, Roth:arXiv]. In certain cases, background scattering from a dense surrounding medium is highly undesirable even for experiments on crystalline samples. For example, imaging techniques have been developed to utilize the faint continuous diffraction signal in regions between and at scattering angles beyond the intense Bragg reflections due to lattice disorder [Ayyer:Nature530:202, Chapman:JAC50:1084] or lattice truncation [Spence:OE19:2866, Kirian:PRX5:011015]. The advantage and motivation for such approaches is that the continuous diffraction that can be accessed provides a direct route to solving the crystallographic phase problem without the need for prior knowledge or additional measurements.

Here, we demonstrate high-resolution x-ray diffraction from isolated protein nanocrystals delivered into the XFEL focus via a convergent-nozzle aerosol injector (CNAI) [Kirian:SD2:041717]. We show that the aerosol delivery produces extremely low background scattering signals compared with a conventional liquid jet. This aerosol injector has essentially the same size and form as the nozzles that are commonly used to produce liquid jets for SFX experiments [DePonte:JPD41:195505, Beyerlein:RSI86:125104, Oberthuer:SR7:44628] and hence can be installed using standard liquid-jet mountings available at x-ray facilities. As shown in our previous work, CNAIs can produce aerosolized beams of sub-micrometer particles with a full-width at half maximum diameter \text$<\unit{5}{\micro\metre}$ and particle velocities on the order of a few hundred meters per second, depending on particle size and operating conditions. This high velocity may be well-suited to the MHz repetition rates of upcoming XFEL sources.

2 Experimental methods

This proof-of-principle experiment was performed on natural Cydia pomonella granulovirus (GV) particles of approximately $200\times 200\times 370~{}\text{nm}^{3}$ in size that consist of a central virus body surrounded by a crystalline granulin protein shell. They infect invertebrates such as the codling moth (Cydia pomonella) [Jehle:VJ346:180]. The GV particles used in this study were purified from a biopesticide solution (Certis Madex HP) using a method described elsewhere [Oberthuer:SR7:44628] and suspended in water at a concentration of approximately $3\times 10^{11}$ particles/ml prior to injection. The particle concentration was measured using a NanoSight (model LM14C) particle analysis system. The volume of the particle is about $\unit{0.015}{\micro\metre^{3}}$, with about 2/3 of that found as the volume of the crystalline shell [Gati:PNAS114:9], which corresponds to a diameter of approximately 300 nm for a sphere of equivalent volume. Despite their small size, previous SFX experiments recorded diffraction to 2.1 Å resolution from such nanocrystals delivered to the x-ray beam in a gas-focused liquid jet [Gati:PNAS114:9].

Diffraction measurements were performed in the nanofocus chamber at the coherent x-ray imaging (CXI) [Liang:JSR22:514] instrument at the Linac Coherent Light Source (LCLS). The experiment was carried out immediately after a successful liquid-jet experiment [Oberthuer:SR7:44628] without disruption to the x-ray beam. During that earlier experiment the beam focus was optimised by adjusting the Kirkpatrick-Baez (KB) focusing mirrors with the help of analysis of spot imprints on a gold foil. After optimisation the position of the beam was determined by placing a YAG screen in the focal plane and observing optical fluorescence with a fixed in-line microscope with a resolution of a few ${\micro\meter}$. In our experiment, the aerosol beam was initially aligned relative to this reference, and then scanned in position as described below.

The granulovirus suspension was aerosolised using a gas dynamic virtual nozzle (GDVN) [Beyerlein:RSI86:125104] mounted in a cylindrical nebulization chamber as depicted in \autoreffig:jet (a). A GDVN uses gas flow focusing to create a liquid jet with a diameter significantly smaller than the orifice of the nozzle, and which consequently breaks up to form a mist of droplets. The liquid was pressurized to flow from the nozzle at rates between $\unit{2.7}{\micro}$l/min and $\unit{3.5}{\micro}$l/min, producing droplets of about $\unit{2}{\micro\metre}$ diameter at a rate between $11\times 10^{6}\text{~{}s}^{-1}$ and $14\times 10^{6}\text{~{}s}^{-1}$, each containing on average 1.3 nanocrystals. This is equivalent to particle flow rates of $8.6\times 10^{8}$–$1.1\times 10^{9}$ particles/min.

The focusing gas was helium, which was set to a mass flow rate in the range of 10–60 mg/min. The nebulisation chamber had an inner diameter of approximately 40 mm and was 150 mm in length, giving a residence time in the chamber of several minutes, and a helium pressure that stabilised at a value between 100 mbar to 1 bar. Under these conditions most of the solvent evaporated to produce nanocrystals suspended in a humid helium atmosphere [Kirian:SD2:041717]. Drops that contained more than one particle during the initial stage most likely formed clusters of crystals [Cho:Langmuir23:12079, Daurer:IUCrJ4:3]. The aerosol flowed through conductive silicone rubber tubing (Simolex, 6.3 mm inner diameter, 30 cm length), which was coupled to a standard “nozzle rod” of the CXI beamline, which is a 1.2 m long stainless steel tube with a 6.3 mm inner diameter that is normally used to transfer liquid-jet injectors in and out of the main experimental chamber without breaking vacuum [Weierstall:RSI83:035108]. The conductive tubing along the entire particle path acted as a Faraday cage to shield external electric fields from interacting with particles that might become charged through triboelectric effects in the GDVN. The aerosol finally exited the CNAI, which was mounted at the end of the CXI nozzle rod much like a typical liquid-jet nozzle. It consisted of a ceramic injection-molded tube of 1 mm outer diameter, $\unit{500}{\micro\meter}$ inner diameter, a short converging section with a convergence angle of 15*∘*, and a $\unit{100}{\micro\meter}$ exit aperture (further details can be found in our previous work [Kirian:SD2:041717]).

During the diffraction experiment we monitored the crystal injection through direct optical imaging of scattered laser light from injected particles [Awel:OE24:6507]. A pulsed Nd:YLF laser (527 nm, $\sim$3 mJ per $\sim$150 ns pulse, 120 Hz) was focused to a $\sim$0.8 mm spot within the aerosol stream, and scattered light was observed through the in-line microscope available at CXI (Questar long distance microscope, model: QM-1 MK III, NA = 0.05 at 750 mm objective distance). Images were recorded using an OPAL-4000 CCD camera and stored at 30 Hz. \autoreffig:jet (c) shows a 3.7 min time-averaged optical image of particles exiting the injector. We determined that particles moved at speeds of approximately 300 m/s when they exited the injector, to arrive at the x-ray interaction point within a flight-time of less than $\unit{1}{\micro\second}$. This particle speed was evaluated from the streak length of recorded particle images produced by laser illumination with a known pulse duration, conducted during laboratory characterization of the CNAI (see \autoreffig:GV (a) and \autorefsec:characterization). The CNAI tip is seen to the left of \autoreffig:jet (c), and the approximate x-ray focal point is indicated by the star. The particle stream could not be observed at points close to the CNAI tip because direct scattering from the tip saturated the imaging CCD.

3 Injector characterization and hit-fraction estimates

In order to develop and characterize the operation of the CNAIs we conducted tests of both 15*∘* and 30*∘* CNAIs in our laboratory. The setup differed from our previous work [Kirian:SD2:041717] by the inclusion of a narrow particle transport tube intended to replicate the delivery system used at CXI. Aerosolized GV particles were transported from the nebulization chamber to the CNAI tip using stainless steel tubing of 4 mm inner diameter and 700 mm length. The GDVN was operated at flow rates of $\unit{2.7}{\micro}$l/min and 28 mg/min for liquid sample and helium, respectively. A GV concentration of approximately $1.6\times 10^{9}$ particles/ml was used (this was diluted by a factor of 200 from the solution that was used at the CXI experiment). This flow rate and sample concentration corresponds to the generation of drops at a rate of approximately $1.1\times 10^{7}\text{~{}s}^{-1}$ and an entrance rate of aerosolized particles of $7.2\times 10^{4}\text{~{}s}^{-1}$.

The imaging setup used for visualizing particles was described in detail previously [Awel:OE24:6507]. Briefly, it was comprised of a Nd:YLF laser (Spectra Physics Empower ICSHG-30, 527 nm, approximate pulse duration 100 ns, repetition rate 1 kHz, pulse energy 20 mJ) to illuminate particles, a high-frame-rate CMOS camera (Photron SA4) and a 5$\times$ magnification, 0.14 NA microscope objective to record images. The laser beam was collimated to a 2 mm spot, such that it illuminated particles across the entire field of view of the camera. The camera exposure time was set to 20 ms, such that each frame contained 20 pulses of the 1 kHz Nd:YLF laser illumination. A single image of particles emerging from the CNAI is shown in \autoreffig:GV (a). The images are streaked due to the high velocity of the particles, and the observed intensity profile of these streaks reflects the relatively fast rise and slow decay of the Nd:YLF laser pulse. Centroid positions of individual particle streaks contained in 23,500 frames were used to produce the rate-corrected two-dimensional particle density map shown in \autoreffig:GV (b). This rate-corrected density has units of particles per area per particle generation rate and is defined as

[TABLE]

where $N_{p}$ is the average number of particles that fall within a spatial bin of area $A$, and $R$ is the rate at which particles entered the injector. Note that $N_{p}$ represents the average particle counts at an instant in time and not a time-integration over many exposures, which is appropriate because we intend to use the particle injector with femtosecond pulses. In our case, $N_{p}$ was computed by summing the number of particles that fell within each spatial bin, and then dividing by the number of recorded images and the number of laser illumination pulses per image.

The measured rate-corrected density $D$ may be used to estimate the optimal hit fraction that could be achieved under idealized conditions in our x-ray measurements. If an entrance rate of $R_{X}$ is used in the x-ray measurements, the 2D particle number density is $DR_{X}$. We define the effective cross-sectional area $\sigma$ such that the average number of particles intercepted by an x-ray pulse is $\sigma DR_{X}$. Assuming Poisson statistics, the probability of intercepting just one particle in an x-ray pulse is

[TABLE]

where the approximation holds to within $\sim$10% error as long as $\sigma DR_{X}<0.1$. We define the x-ray beam diameter as $d_{X}$ and the particle beam diameter as $d_{p}$ and estimate two limiting cases for the effective cross sectional area. The first case, $\sigma^{+}=\frac{\pi}{4}(d_{x}+d_{p})^{2}$, describes the optimistic limit in which a particle at the periphery of the x-ray beam produces acceptable diffraction. The second case, $\sigma^{-}=\frac{\pi}{4}(d_{x}-d_{p})^{2}$, corresponds to the stronger assertion that an acceptable diffraction pattern requires that the entire x-ray-beam width falls within the particle (if $d_{X}<d_{p}$) or that the entire particle falls within the x-ray-beam width (if $d_{p}<d_{X}$). Finally, we arrive at two limiting hit-fraction estimates:

[TABLE]

The maximum rate-corrected particle density recorded in the laboratory, i. e., at the focus of the particle beam shown in \autoreffig:GV (b), was $D\approx{\unit{2.2\times 10^{-9}}{\micro\meter}}^{-2}\text{~{}s}$. Assuming the approximate values $d_{X}\approx 150$ nm, $d_{p}\approx 300$ nm, and $R_{X}\approx 11\times 10^{6}\text{~{}s}^{-1}$ suggests that the maximum hit fraction to be expected in our XFEL diffraction measurements is in the range $H_{1}^{-}\approx 0.04$ % to $H_{1}^{+}\approx 0.4$ %. This predicted hit fraction is much higher than the hit fraction we achieved during the CXI experiment, as discussed in the next section.

4 X-ray diffraction analysis and discussion

Diffraction measurements were conducted at a photon energy of 8 keV and an estimated average pulse energy of 4.2 mJ prior to the $\sim$30-50 % beamline transmission losses [Boutet:2016:pricom]. The CSPAD detector was located 127.9 mm downstream from the x-ray focus. We recorded detector data frames for every X-ray pulse, at a rate of \unit120\hertz, for a cumulative total of 1.3 hours, which resulted in approximately 560,000 data frames.

In all of our diffraction analysis we excluded all pixels from each detector frame that had abnormally high or low variances or mean values in “dark” measurements made without x-rays, as well as a few patches of pixels for which there was obvious stray-light background. For every frame, the dark measurement was subtracted, and then a uniform common-mode electronic noise constant was subtracted from each detector panel. The common-mode offset was determined from unbonded detector pixels that are not sensitive to x-rays. The detector gain relating detector digital units to photon counts per pixel was obtained from a histogram of the pixel values, which yielded clear peaks corresponding to counts of zero, one, and two photons. Most of this analysis was performed using the Python psana package provided by LCLS [Damiani:IUCrJ49:672].

\autoref

fig:gv-aerosol shows one quadrant of a recorded diffraction pattern from an aerosolized GV crystal, where the average detector dark frame and common-mode offsets have been subtracted. A total of 33 hits from GV were recorded, corresponding to a hit fraction of $\sim\!0.006$ %. 24 patterns (73 % of hits) were indexed using the CrystFEL software suite [White:JACR45:335]. Autoindexing failed on patterns that appeared to consist of multiple crystals clumped together. We expect the hit fraction for our aerosol injector to be significantly lower than a typical liquid jet (about 1-10%) because of the $\sim$25-fold higher particle speed of the aerosol beam and the $\sim$4-fold reduction in liquid flow rate. However, our recorded hit fraction was still lower than the range $0.04-0.4$ % that we estimated from our laboratory measurements.

For comparing the background obtained using the CNAI to that typically observed in liquid-jet experiments we examined data from a previous SFX experiment [Oberthuer:SR7:44628] in which the exact same GV sample was injected into the x-ray beam as a liquid suspension with a GDVN. All experimental parameters were identical in both the CNAI and GDVN measurements except for the pulse energy, which was 4.6 mJ on average for the GDVN measurements.

The comparison of background scattering for the CNAI and GDVN approaches is presented in \autoreffig:background, which shows a plot of the normalized azimuthally-averaged profiles of scattered-photon counts (per pixel and per mJ of pulse energy), as a function of photon-wavevector transfer. The per-pixel standard deviations in the measurements are indicated by the gray regions in \autoreffig:background. The average profiles were divided by the average pulse energy to account for the slightly higher pulse energy in the case of the GDVN. The frames used in \autoreffig:background were sampled uniformly from the final $\sim$5 minutes of data collection, when the conditions were closest to optimal, although little difference was noticed in other measurement segments. We excluded frames that fell below 1 mJ pulse energy. We additionally excluded frames that were visually corrupt as well as those for which the x-rays obviously missed the liquid jet, which corresponded to less than 10% of the frames. After removing these outliers, we confirmed that more than 10,000 frames contributed to each of the two profiles.

As can be seen from the plots in \autoreffig:background, the liquid jet produces a background that is over 1000 times higher at a wavevector transfer of $q=2\sin(\theta)/\lambda=0.32\text{\AA}^{-1}$, corresponding to a resolution of 3.1 Å, where $\theta$ is the Bragg angle and $\lambda$ the wavelength. This coincides with the mean distance between oxygen atoms in water where diffuse scattering from water has its maximum. At low scattering angles the background from the liquid jet was about 200 times higher than for aerosol injection. The liquid jet for these measurements was operating at a flow rate of $\unit{20}{\micro}$l/min. Typical liquid flow rates needed to produce a stable jet range from $\unit{5-30}{\micro}$l/min, depending on the viscosity and surface tension of the liquid and the nozzle geometry. The volume of liquid that interacts with the x-ray beam scales roughly as the square root of the volumetric flow rate [Beyerlein:RSI86:125104], and thus the liquid jet background is rather typical.

Due to our convergent micro-focused particle beam, hit fractions are highly sensitive to the relative positioning of the CNAI with respect to the x-ray beam. Our initial diagnostic for particle beam positioning was direct imaging of scattered light, which allowed for the rough positioning of the CNAI. From this initial position, it was necessary to perform a subsequent two-dimensional scan of the injector position in an effort to optimize the spatial overlap between particle beam focus and x-rays. Due to the limitations of our 6-hour measurement shift, we only performed one $\unit{200}{\micro\meter}$ scan in the direction transverse to the particle beam and one $\unit{400}{\micro\meter}$ scan along the particle beam direction. It is therefore highly unlikely that we located the ideal position that maximizes the hit fraction. However, we expect that the background scatter we observed is representative of the gas and water vapor exiting the injector because the gas expansion into vacuum is highly divergent. Direct imaging of the gas density leaving the CNAI [Horke:JAP121:123106] shows that the gas plume spans volume hundreds of micrometers wide around the XFEL beam position.

Another possible culprit for our sub-optimal hit fraction is a sub-optimal aerosol transmission efficiency, which might be remedied by reducing the overall transportation tube length, increasing the particle generation rate, decreasing the particle speed, increasing the volumetric flow rate of carrier gas, or by the addition of aerodynamic lenses within the transport tube, which would maintain particles near the center of the transport tube. Although aerosol injection hit fractions tend to be relatively low in comparison to liquid jets, recent work at at the CXI instrument reported hit fractions of $0.83~{}\%$ for aerosolized 40 nm viruses delivered with an aerodynamic lens stack aerosol injector [Daurer:IUCrJ4:3].

Although it is convenient that our miniaturized CNAI is compatible with standard GDVN mounting hardware, the downside is that the small exit aperture, $\unit{100}{\micro\meter}$ diameter in our case, is prone to clogging. We have successfully operated our CNAIs in the laboratory for many hours without interruption, but clogging typically occurs whenever the aerosolization liquid jet misbehaves and produces large droplets for a period of a few minutes. It is, therefore, essential to ensure the formation of small droplets and continuous flow of carrier gas. In the XFEL experiment reported here, there were a total of three clogged aerosol-nozzles, each of which required $\sim$20-30 minutes to replace. The severity of this issue could be greatly reduced by filtering out large droplets with, for example, an inline impactor [Maenhaut:NIM109:482], and by using electrospray ionization to produce smaller initial droplet diameters [Yamashita:JPC88:4451, Chen:JAS26:963, Bogan:NanoLett8:310].

It must finally be noted that the GV crystals utilized here are notoriously robust and survive in nearly pure water. For crystals that dissolve, for instance, upon varying pH, it may be feasible to avoid droplet evaporation by using a humidified carrier gas, by using electrospray nebulization, or by simply placing the nebulization source close to the entrance of the aerosol nozzle to reduce the time of transport.

5 Conclusions

We demonstrated x-ray diffraction from aerosolized sub-micrometer protein crystals with background levels drastically lower than in typical SFX experiments utilizing liquid jets. This may be important for coherent-diffractive-imaging experiments on weakly scattering targets such as isolated proteins, viruses, or cells, as well as for the measurement of diffuse scattering or lattice-transform signals between crystalline Bragg reflections [Ayyer:Nature530:202, Kirian:PRX5:011015]. We showed that our injector is compatible with the existing hardware at LCLS, allowing quick changes from a liquid jet to an aerosol injection system in a single experiment. The relatively high ($\sim$300 m/s) particle velocities may be useful for avoiding damage due to x-ray induced explosions when using new XFEL sources with pulse repetition rates up to 4.5 MHz.

While the obtained 0.006% hit fraction at LCLS was much lower than in typical liquid jet x-ray diffraction experiments, laboratory measurements suggest that this can be improved by orders of magnitude. Based on these laboratory measurements, we suspect that the low hit fractions observed in this study are a result of aerosol transport losses, clustering of particles, clogging of the aerosol-nozzle due to an under-performing GDVN nebuliser, or misalignment between the x-ray focus and particle beam focus. As we have noted, there are several possible routes to improve upon the injection strategy described here, as evidenced by other aerosol injection work performed at the same CXI instrument [Daurer:IUCrJ4:3].

Above all, the lower background achieved with the aerosol nozzle somewhat offsets the lower hit fraction, since the number of required measurements depends inversely on the square of the signal to noise ratio of intensities, or directly proportional to the background counts.

This proof of principle experiment was performed on granulovirus occlusion bodies suspended in water. These protein crystals have naturally evolved to be robust against the change in the buffer conditions and dehydration caused by evaporation of the liquid layer on the crystals surface. However, most protein crystals are not stable in pure water. When working with other types of crystals, the liquid buffer evaporation rate on the surface of the crystals must be controlled, for example by controlling the relative humidity at the crystals [Sanchez-Weatherby:ACD65:1237, Roedig:SR5:10451].

\ack

Acknowledgments

We thank the LCLS staff for accommodating laser-light-scattering imaging in the CXI instrument during our experiments. Portions of this research were carried out at the LCLS at the SLAC National Accelerator Laboratory. This LCLS beamtime was part of the Protein Crystal Screening (PCS) program. LCLS is an Office of Science User Facility operated for the US Department of Energy Office of Science by Stanford University. Use of the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, is supported by the U. S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515. Parts of the sample delivery system used at LCLS for this research was funded by the NIH grant P41GM103393, formerly P41RR001209.

In addition to DESY, this work has been supported by the excellence cluster “The Hamburg Center for Ultrafast Imaging–Structure, Dynamics and Control of Matter at the Atomic Scale” of the Deutsche Forschungsgemeinschaft (CUI, DFG-EXC1074), the Gottfried Wilhelm Leibniz Program of the DFG, the European Research Council under the European Union’s Seventh Framework Programme (FP7/2007-2013) through the Synergy Grant AXSIS (ERC-2013-SyG 609920) and the Consolidator Grant COMOTION (ERC-Küpper-614507), the Helmholtz Association “Initiative and Networking Fund”, and the Australian Research Council’s Discovery Projects funding scheme (DP110100975). R.A.K. acknowledges support from an NSF STC Award (1231306).

\referencelist

[aerosol-sfx]