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Necessary Experimental Conditions for Single-Shot Diffraction Imaging of DNA-Based Structures with X-Ray Free-Electron Lasers Zhibin Sun, Jiadong Fan, Haoyuan Li, Huajie Liu, Daewoong Nam, Chan Kim, Yoonhee Kim, Yubo Han, Jianhua Zhang, Shengkun Yao, Jaehyun Park, Sunam Kim, Kensuke Tono, Makina Yabashi, Tetsuya Ishikawa, Changyong Song, Chunhai Fan, and Huaidong Jiang ACS Nano, Just Accepted Manuscript • DOI: 10.1021/acsnano.8b01838 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 11, 2018

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Necessary Experimental Conditions for Single-Shot Diffraction Imaging of DNA-Based Structures with X-Ray Free-Electron Lasers Zhibin Sun,1,2,3 Jiadong Fan,3 Haoyuan Li,2,4 Huajie Liu,5 Daewoong Nam,6,7 Chan Kim,8,9 Yoonhee Kim,8,9 Yubo Han,10,11 Jianhua Zhang,1,3 Shengkun Yao,3 Jaehyun Park,6 Sunam Kim,6 Kensuke Tono,12 Makina Yabashi,13 Tetsuya Ishikawa,13 Changyong Song,7 Chunhai Fan,5 and Huaidong Jiang3,* 1

2

State Key Laboratory of Crystal Materials, Shandong University, Jinan 250100, China

Linac Coherent Light Source, SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, USA

3

School of Physical Science and Technology, ShanghaiTech University, Shanghai 201210, China 4

5

School of Physics, Stanford University, Stanford, California 94305, USA

Laboratory of Physical Biology, Shanghai Institute of Applied Physics, Chinese Academy of Science, Shanghai 201800, China

6

Pohang Accelerator Laboratory, Pohang University of Science and Technology, Pohang 37673, Korea


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Department of Physics, Pohang University of Science and Technology, Pohang 37673, Korea 8

9

European XFEL GmbH, Holzkoppel 4, Schenefeld 22869, Germany

Department of Physics and Photon Science & School of Materials Science and Engineering, Gwangju Institute of Science and Technology, Gwangju 61005, Korea 10

Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China

11

SLAC National Accelerator Laboratory, 2575 Sand Hill Road, Menlo Park, California 94025, USA

12

Japan Synchrotron Radiation Research Institute, Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5198, Japan 13

RIKEN SPring-8 Center, Kouto, Sayo-cho, Sayo-gun, Hyogo 679-5148, Japan

*E-mail: [email protected] ABSTRACT: It has been proposed that the radiation damage to biological particles and soft condensed matter can be overcome by ultrafast and ultra-intense X-ray free-electron lasers (FELs) with short pulse durations. The successful demonstration of the “diffraction-beforedestruction” concept has made single-shot diffraction imaging a promising tool to achieve high resolutions under the native states of samples. However, the resolution is still limited because of the low signal-to-noise ratio (SNR), especially for biological specimens such as cells, viruses, and macromolecular particles. Here, we present a demonstration single-shot diffraction imaging experiment of DNA-based structures at SPring-8 Angstrom Compact free electron LAser (SACLA), Japan. Through quantitative analysis of the reconstructed images, the scattering


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abilities of gold and DNA were demonstrated. Suggestions for extracting valid DNA signals from noisy diffraction patterns were also explained and outlined. To sketch out the necessary experimental conditions for the three-dimensional (3D) imaging of DNA-origami or DNA macromolecular particles, we carried out numerical simulations with practical detector noise and experimental geometry using the Linac Coherent Light Source (LCLS) at the SLAC National Accelerator Laboratory (SLAC), USA. The simulated results demonstrate that it is possible to capture images of DNA-based structures at high resolutions with the technique development of current and the next-generation X-ray FELs facilities.

KEYWORDS: DNA-origami, X-ray free-electron laser, single particle imaging, coherent diffraction imaging, X-ray diffraction. The advent of X-ray FELs

1, 2

determination of the ultrasmall structures of biological particles, functional materials.

7

3

and

5, 6

and

enabled the capture of ultrafast dynamic processes 4

protein crystals,

To capture the inner structures of biological particles such as cells,

viruses, and organelles at the atomic resolution, radiation damage has to be taken into serious consideration. 8 Because of the tunable pulse durations of X-ray FELs, the photoelectric effects that lead to radiation damage can be reduced or mitigated by short pulses < 10 fs. 9 Based on the innovative “diffraction-before-destruction” concept, cells,

11, 12

viruses,

4, 13

organelles,

14, 15

10

nanoparticles,

single-shot diffraction imaging of living 16-19

and aerosol particles

20, 21

without

observable damage has been achieved. To realize the full potential of coherent diffraction imaging methods with X-ray lasers, the Single-Particle Imaging (SPI) Initiative

22

has, since

2015, devoted numerous studies to the radiation damage problem, development of X-ray FEL instruments, sample and delivery issues, phase retrieval algorithms, and development of data


3

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analysis software.

23-26

Recent results have demonstrated 3D imaging of the Rice Dwarf Virus

and PR772 bacteriophage at a resolution of about 10 nm,

27, 28

which is a great step-by-step

approach to the atomic resolution. Apart from the radiation damage issues and limited resolution 29

of single-shot diffraction imaging,

the diffraction efficiency of biological particles is also an

obstacle. The coherent X-ray scattering cross-section of Low-Z elements is lower compared to electrons.

30, 31

The methods by which to enhance the SNR and extract valid signals from noisy

patterns are still challenging. The past decade has witnessed a dramatic growth in DNA assemblies and extraordinarily powerful scaffolded DNA origami.

32

State-of-the-art user-defined dense 3D scaffolds are being

used for molecular transport, enzymatic catalysis, and drug delivery system designs.

33-35

In

contrast, fabricating gold nanoparticles (AuNPs) with DNA strands is a powerful tool for controllable self-assembly, DNA-based plasmonic nanostructures, and well-defined moleculelike architectures.

36, 37

Cryo-EM

38

and atomic force microscopy (AFM) 39 are the general tools

employed to determine the structures of 3D scaffolds and self-assembled nanostructures. Singleshot diffraction imaging of single-molecules and single-particles with X-ray lasers have been proved to be another powerful idea to structure determination.

40-42

However, up to now, taking

snapshots of DNA molecules, particles, and assembled structures with X-ray FELs is still absent. 43

In this study, we have attempted to determine the structures of DNA-based plasmonic nanoparticles by single-shot diffraction imaging with X-ray FELs. The diameter of AuNPs used for this research is larger than the width and thickness of DNA chains. Groups of 2D diffraction patterns were collected within a few minutes. Effective signals were extracted from noisy patterns and the associated AuNPs were phased to real-space images. To determine the different


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performances of gold and DNA under 7 keV of photon energy, the experimental diffraction intensity combined with the coherent scattering cross-section, Mie scattering theory, and Porod’s Law were used for further analysis. In addition, the 3D imaging of reproducible biological particles has attracted growing interests from the scientific community. To provide some references for the single-particle imaging of DNA origami scaffolds, numerical simulations with the experimental parameters used by the SPI Initiative at the LCLS, SLAC, were implemented. The successful simulated trial of the 3D icosahedral scaffolds demonstrated that it is feasible to capture single-shot images of user-defined biological particles under current experimental conditions. EXPERIMENT AND DATA ANALYSIS The experiments were performed at Beamline 3, SACLA, Japan. The experimental setup is illustrated in Figure 1. X-ray laser pulses with a photon energy of 7 keV were focused to a spot size of approximately 1.5 µm by a pair of Kirkpatrick-Baez (KB) mirrors. The focused beam spot size was determined by a knife-edge scan with Au wire at the sample position. A pulse picker was used to reduce the repetition rate of X-ray laser pulses from 10 to 1 Hz. The pulse duration was set to about 10 fs. The single-shot pulse energy was evaluated to be about 100 µJ and the intensity fluctuation of pulses in 30 shots (standard deviation) was approximately 8.8%. To block parasitic scattering from the KB mirrors and apertures upstream, a four-way cross slit with a bevel-cut and micro-polished edge was applied. Monodispersed DNA-based AuNP dimers composed of two 50-nm diameter AuNPs and single-stranded DNA origami were obtained by chemical synthesis (The detailed synthesis procedures are provided in the Methods section. Figure S1 in the Supporting Information). AuNP dimers were dispersed onto the Si3N4 membrane chip along with an alcohol solution and were air-dried. Then, the 100-nm-thick


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membrane (with a 36×36 multi-window array) was mounted on a goniometer in a MultipleApplication X-ray Imaging Chamber

44

under a vacuum atmosphere. By raster scanning across

the Si3N4 membrane, diffraction patterns were collected by a Multi-Port Charge-Coupled Device (MPCCD) detector located about 1500 mm downstream of the sample stage.

Fig. 1. Schematic of single-shot diffraction imaging layout with X-ray FELs at SACLA. The Xray FEL pulses are focused to about 1.5 µm by a pair of KB mirrors. After passing through the slits, X-ray lasers interact with the DNA-based AuNPs. By demonstrating the zig-zag raster scan, fresh samples can be delivered to the X-ray laser beam path. To remove the artifacts from the raw data, different backgrounds were collected periodically. Parts of the backgrounds were captured with no photons (dark noise), which can be used to reflect the different performances of multiple readout channels of the MPCCD detector over time. Other backgrounds were determined from non-hit patterns. These patterns can be used to subtract incoherent scattering from the X-ray FEL instruments and optics, and unwanted scattering from the membrane. The background was defined by counting the number of pixels containing values below the predetermined analog-to-digital unit (ADU) threshold. To obtain a


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rapid and rough estimation of the hit rate by the fixed-target sample delivery method, 792 coherent diffraction patterns collected in approximately 20 min were analyzed. We found that at least 60 diffraction patterns could be marked as valid far-field diffraction data. These data contain single, two, three, and multiple gold NPs. Supervised pattern classification was applied to sort the data after background subtraction. The classification (Figure S2 and descriptions in the Supporting Information) was based on the appearance of speckles within the low-frequency region. Taking advantage of this visual method, the target dimer structures were recognized. The Si3N4 membrane gathers dirt during the sample preparation. In addition, the focused beam spot size is large compared to the DNA-based AuNPs. These lead to noisy far-field diffraction patterns. To improve the SNR and make the reconstruction feasible, 7×7 pixels were added into one pixel. Figure 2 presents three representative diffraction patterns and the related reconstruction images. The diffraction patterns shown here have been extended to the edge of the detector after 7×7 downsampling. Hybrid input–output (HIO) 45 and guided hybrid input–output (GHIO)

46

phase retrieval algorithms were used for the phasing. Other commonly used

algorithms, such as oversampling smoothness

47

and relaxed averaged alternating reflections,

48

were also used. There is no clear difference under different phase retrieval algorithms (Figure S3 in the Supporting Information). To eliminate the influence of random phases, 100 independent reconstructions were implemented. The final image is the average taken from the best 16 results (Figure S4 in the Supporting Information) with the minimum error. Distinct light shadows around the reconstructed particles originate from the dirty Si3N4 membrane.


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Fig. 2. Single-shot diffraction patterns and associated phasing results. Diffraction patterns of (a) two, (b), three, and (c) multiple particles sorted during the data analysis processes. To improve the single-to-noise ratio, 7×7 pixels were binned into one pixel. The size of the diffraction patterns presented here is 300×300 pixels. The spatial frequency was determined by momentum transfer q 2sinθ⁄λ, where θ is half of the diffraction angle and λ is the incident wavelength. To determine the real images, HIO and GHIO phase retrieval algorithms were applied for (d) two, (e) three, and (f) multiple particles. The patterns were shown in natural logarithm scale and the bars represent 50 nm.


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Fig. 3. Simple quantitative analysis and electronic images of AuNPs. (a) Fast histogram of diameter distributions of part of the reconstructed AuNPs. The mean diameter is approximately 51 nm, close to the known size of the AuNPs (50 nm). (b) Center-to-center distance of dimeric AuNPs. The maximum frequency of the gap between AuNPs is about 70 nm, equal to the designed value. (c) AFM and (d) TEM images of dimeric AuNPs. Capturing images of DNA chains is our main target for this demonstration experiment. However, only AuNPs could be detected from the phasing results. It is important to determine what happened to the DNA chains. Thus, the experimental parameters, data analysis, and phasing processes were investigated; nothing invalid was found. Figure 3 presents the doublecheck results of the reconstructed AuNPs by simple statistical analysis and electronic images. The statistical data come from a small part of the dataset (see Supporting Information Figure S5), but the behavior could still reflect the total truth. The diameter and center-to-center gap between nanoparticles are almost the same as what we know from our sample, as confirmed in Figs. 3(a)


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and 3(b). Figures 3(c) and 3(d) show the AFM and SEM characterizations of our sample. It’s noteworthy that the orientation of the target DNA-based AuNP dimers could cause variations in the gaps between two NPs. Orientation determinations and statistical analysis of the data have to be complemented if accurate gap values are required. 49 Continuing to determine how the DNA chains changed, the Si3N4 membrane used during the experiment was investigated by optical microscopy, as shown in Fig. 4(a). The holes are slightly larger than the focused X-ray spot. Dirty water stains can be seen around the holes. These are light shadows around the reconstructed AuNPs, as mentioned above. Quantitative analysis was conducted for the diffraction signals and background. Figure 4(b) presents the average radial intensity of the dimer, trimer, and multiple particles and the background. The signal of the background is equal to the dimer and trimer when the momentums transfer Q drops to about 48 µm–1, corresponding to a full spatial resolution of about 13 nm. For multiple particles, the scattering intensity should be stronger than the dimer and trimer. However, weaker signals are observed from the curve. In addition, the intensity of the associated reconstructed AuNPs in Fig. 2(f) exhibits a non-uniform electron density distribution among AuNPs. This may originate from the relative position between the X-ray laser beam and the particles. In other words, the particles were illuminated by different parts of the X-ray FEL Gaussian beam. A similar phenomenon could also be observed in other phasing results. 50


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Fig. 4. Scattering ability of gold and DNA. (a) Optical image of the Si3N4 membrane after the interaction with XFEL pulses. Colorful regions and water stains originate from the sample preparation and buffer solution. (b) Power spectral density of AuNPs and DNA. The diffraction intensity of the background is close to that of dimeric AuNP after 0.96 nm–1, corresponding to a full-period resolution of 13 nm. Here, the momentum transfer q was determined by 4 ⁄. (c) Coherent scattering cross-section of gold and DNA. The scattering efficiency of gold is about 16 times stronger than that of DNA under 7 keV. (d) Theoretical calculations of scattering intensity of gold and DNA. The calculations were based on Mie theory and Porod’s law. The maximum intensity of each peak for gold is about 100 times stronger than that for DNA. The DNA signals are unobservable because of the ultra-intense background noise. At the same time, the scattering ability of gold and DNA (C10H11N4O6P) is different; Fig. 4(c) demonstrates this difference. For gold, the coherent scattering cross-section is about 6.5 cm2/g under 7 keV, but for DNA, it is about 0.4 cm2/g. This vast difference leads to different diffraction efficiencies


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and performances. Figure 4(d) presents the scattering intensity calculated by Mie theory and

Porod’s Law. The scattering intensity is expressed as ∝ . By solving Maxwell’s equation for spheres of arbitrary size (Mie theory) with the appropriate boundary conditions, the resulting scattered far-field components of a point (r, θ, φ) in spherical coordinates for a unit-amplitude incident field can be written in the following form:

#

%$

#

%$ − ! "

&' () (),

(1)

(). (),

(2)

0#.

230 0 (&' ) + 50 60 (&' )7,

(3)

0#.

250 0 (&' ) + 30 60 (&' )7,

(4)

−- − ! " with ∞

). () ∑01. ∞

) () ∑01.

0(0#.)

0(0#.)

where Eθ is the scattered far-field component in the scattering plane, defined by the incident and scattered directions, and Eφ is the orthogonal component. : ( 2⁄) is the propagation constant (wave number) in vacuum and is the wavelength. ! ;% is the time variation, 30 , 50 are coefficients determined from boundary conditions, and 0 , 60 are functions of the scattering angle. Detailed theory can be found in Ref. [51]. 51 The particle size, concentration, polarization, refractive index, and angle are the main parameters for Mie theory. To guarantee the comparability, the calculations for gold and DNA were conducted under the same condition. The photon energy was 7 keV and the beam mode was set to a plane wave instead of a Gaussian beam, to eliminate the influence of the beam position and guarantee the same illumination flux. The mass densities of gold and DNA were 19.32 g/cm3 and 1.70 g/cm3, 52 separately. The complex refractive index of gold and DNA under


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7 keV was set to be 1−6.25×10–5 − i·8.01×10–6 and 1−7.46×10–6 − i·4.91×10–8, respectively.

53

After the calculation, we found that the scattering intensity of gold was almost 100 times larger than that of DNA under the same momentum transfer. To verify the calculation, Porod’s Law that is usually used for small-angle X-ray scattering (SAXS) was employed to the peak fitting. 54 (? ∆A|∆C| D4

E0(FG)"FGHIE(FG) GJ

MNO (? ∆A|∆C| 16 Q < "R,

K ,

(5) (6)

where I0 is the incident intensity, Imax is the maximum ring intensity, re is the classical electron radius, ΔΩ is the solid angle, ∆C is the difference of the complex electron density of the sphere and the medium, q is the momentum transfer determined by 4 ⁄ , and R is the sphere radius. The well-fitted lines demonstrate that the scattering efficiency and ability between gold and DNA are significant. In this study, spherical particle models were used for the calculations and fitting. As we all know, different particle size, shapes, and their correlations cause diverse scattering intensities. Thus, appropriate models should be used. To step closer to the practical conditions, golden-core and DNA-shell models were created. The intensity difference between golden-core and DNA-shell (Figure S6 and the details of the calculation were provided in the Supporting Information) remains the same level as particle models demonstrated. These constitute the reasonable reasons for the “disappearance” of the DNA chains under noisy conditions. SIMULATION AND DISCUSSION With the above analysis, we can conclude that the reason for the “disappearance” of DNA chains in our final results can be divided into two parts. The first is the sample preparation procedure. To make the Si3N4 membrane more wettable, the membrane must be charged. However, this


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helps not only the solution but also the contaminants to spread onto the membrane quickly. The dirty membrane will increase the background and limit the SNR of the DNA. Although the buffer solution could be diluted and cleaned by centrifuge, the sample solution is still difficult to spread across the membrane. The concentration and monodispersity are difficult to control, especially for some biological samples such as Staphylococcus aureus and mitochondria. In addition, it is more difficult to guarantee the density of single particles or single structures compared to particle injectors. From the behavior of SPI,

40

it is better to use particle injectors

such as gas dynamic virtual nozzles and aerodynamic lens stacks. For particle injectors, the hit rate and gas background may be a problem. Nevertheless, higher X-ray FEL repetition rates could be used to improve the utilization rate of the beamtime. More raw data could be recorded within the same duration compared to low X-ray laser repetition rates. The disadvantage is that it is difficult for some samples to form droplets. In our case, the maximum operating rate of SACLA is 60 Hz, the use of a fixed target is compatible with the repetition rate, and the sample consumption of the fixed target is low compared to particle injectors. In addition, no artifacts will come from the fluctuations in focal position. For fixed targets, the membrane is aligned perpendicular to the X-ray laser beam. However, the strong background negates all advantages. In other words, for weak scattering targets such as bioparticles, the particle injector is the more reasonable choice. The second reason is the sample itself. The maximum size of the DNA-based AuNP structure is about 210 nm. The diameter of AuNPs is about 50 nm. However, the thickness and width of the single-stranded DNA chains used here are 2 and 20 nm. This is challenging under the current development of technology. The smallest 3D spatial resolution of biological particles currently reported by the SPI team is approximately 10 nm. 27, 28 The DNA chains could not be recognized


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clearly under the current resolution. In addition, the DNA is composed of low-Z elements. Regarding the size and scattering ability difference between gold and DNA, it is easy to understand the poor performance of DNA strands. The requirements of the X-ray FEL beamtime are increasing more rapidly. Nevertheless, the beamtime supplied across the world is still limited. To add value to our above demonstration experiment and outline the necessary experimental conditions toward the future single-shot diffraction imaging of DNA-based particles and structures, we conducted numerical simulations with experimental parameters at the LCLS. A realistic simulation of the diffractive experiment requires practical detector effects, geometric corrections, and a clear differential scattering section. It is impossible and futile to obtain an accurate differential scattering section with current techniques. Therefore, several approximations are utilized. Currently, the resolution of SPI is far from the final atomic resolution goal. In addition, no molecular effects are necessary, as they will not have detectable effects on the patterns. According to quantum electrodynamics, regarding the first-order Feynman diagrams, when the polarization states of photons are decoupled from the interaction of the photons with a local source, the differential scattering cross-section of the local source is E − : _ `[A abE |b c ? [\ ]E ^:

h `∙j de^f g df

(k)J

abE |b c ! ∅ &' ()

](l)[l m [A

(7) (8)

where abE |b c is the inner product of the polarization vectors of the scattered and incident photons, [Ω indicates the measure of the unit sphere inherited from canonical Euclidean space R3, and θ is the angle between the polarization of the incident photon and the normal direction of the plane spanned by the incident momentum and scattered momentum of the photon. ϕ is a


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phase factor associated with the two quantum states that have no influence on later calculations. Notice that the length of the scattered photon has the same momentum as the incident photon. This is because the pixel size is small compared to the distance between the interaction point and detector. The solid angle spanned by one pixel can be approximated by

(9)

[A (q/4> )&' s

To obtain the theoretical photon number, shot noise due to the quantum nature of the object should be taken into consideration. E − : _ )vA u ]E (:

(10)

Equation 10 is the mean value of the photons numbers of our measurement if we carry out the analysis infinite times. According to quantum optics, the photon number obeys the Poisson distribution with the mean value described by Eq. 10. Thus, by generating a Poisson random integer according to the theoretical mean value of each pixel, we obtain an ideal photon pattern. By adding random noise, shot noise, solid angle corrections, and polarization corrections to the ideal photon pattern, the noise pattern can be finally obtained. Figure 5 presents the 3D simulations of DNA origami icosahedra and Rice Dwarf Virus (RDV) with no noise. The 3D DNA origami scaffolds were generated by the online open-source DAEDALUS software, provided by the Laboratory of Computational Biology & Biophysics at MIT

55

. The length of each edge is 94 base pairs, equal to about 31 nm. To attach the practical

effects to the simulated data, the geometry of pnCCD was applied during the theoretical calculations. Sample-to-detector distance, central gap, gain were all combined in the geometry file. The detector layout is the same as SPI experiment AMO86615.

26

The pnCCD always used

for SPI experiments at the Atomic, Molecular & Optical Science (AMO) instrument 56 of LCLS. The pixel size of the detector is 75 µm and the quantum efficiency is over 0.8 from 0.3 to 12


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keV. In hard X-ray FELs regime, the photon energy 7.0 keV was used. A detector distance of 1500mm was employed, equals to the SACLA experiment. An ideal focused Gaussian beam with a spot size of 0.1×0.1 μm was used, the same as nanofocusing at Coherent X-ray Imaging (CXI) instrument 57 of LCLS. In addition, the photons per pulse were set to 1.78×1012, equals to 2 mJ/pulse at the sample position. The pulse energy will be 4 mJ at the end of the undulator when a 50% transmission efficiency of the X-ray optics system was assumed. It's compatible with the LCLS high charge mode. For soft X-ray FELs, a pulse energy of 2 mJ was also used at the sample position, equals to a photon number of 7.80×1012. A sample-to-detector distance of 581mm was used, the same as above-mentioned SPI experiment at the AMO of LCLS. The focused beam spot size was 1.5 µm2. Figures 5(a) and 5(b) are 3D diffraction volumes under hard and soft X-ray FELs, respectively. The appearance of speckles is the same. The only difference originates from the oversampling ratio and the spatial frequency. The fine speckles and strong diffraction indicate that the 3D DNA-origami scaffold has excellent five-fold symmetry. Commonly used samples for SPI experiments are solid targets, such as viruses, bacteria, organelles, clusters, and nanoparticles. For DNA origami, the structure is similar to particle shell, that’s to say, the whole atom number is much less than the commonly used interests. The atom number of the DNA origami is 330,143. For RDV, the atom number is 3,487,800. Hereby, it’s better to put a RDV result here as a reference, as shown in Figure 5(c). The photon energy of RDV simulation was set to 2.0 keV, and the pulse energy, beam size, sample-to-detector distance was the same as soft X-ray FELs simulation of DNA origami. For experiment, the principle of photon energy selection was the achievable resolution, diffraction efficiency, and the instrument conditions. To avoid the fluorescence comes from the upstream Silicon apertures, a photon energy below the Silicon K-edge (1.80 keV) is a better. The PDB file


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of RDV was obtained from the Protein Data Bank (PDB ID: IUF2) 58 and the PDB file of DNA origami was shown in Figure 5(d). Hollows of the origami scaffold could be seen everywhere. In order to compare the diffraction intensity, 100 random 2D slices (Figure S7, five representative slices for each diffraction volume were provided in the Supporting Information) were generated from the three diffraction volumes. By summing the 2D slices with random orientations, SAXS patterns were obtained and shown in Figure 5(e), (f), and (g). Compared with the RDV pattern, the diffractive intensity of DNA origami is two orders of magnitude smaller. Figure 5(h) demonstrates this conclusion obviously. To our knowledge, the best spatial resolution for RDV is about 10 nm currently. If the same diffractive intensity is required for DNA origami, then the incident X-ray pulses should be at least 10 times (for hard X-ray FELs) and 50 times (for soft X-ray FELs under our simulated parameters, such as focused beam size, photon energy, and oversampling) stronger than 2mJ at the sample position. This is impossible for current X-ray FEL facilities. The worse thing lies in that it becomes more challenging when backgrounds from particle injectors combined with the diffractive signals. The low SNR also makes data analysis time-consuming and difficult. That’s to say, every single diffractive photons should be employed efficiently during the data processing. At the same time, noise and background reduction technique developments are of great significance for such hollow biological structures.


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Fig. 5. Three-dimensional simulation of 3D DNA-origami icosahedral structure and Rice Dwarf Virus. (a), (b) Three-dimensional diffraction volume of DNA-origami structure with a photon energy of 7.0 keV and 1.6 keV, respectively. (c) Three-dimensional volume of Rice Dwarf Virus under 2.0keV. (d) Three-dimensional structure of DNA-origami. The length of each edge is 94 base pairs (~31 nm). (e), (f), (g) Summation of 100 independent 2D slices with random orientations generated from 3D volumes shown in (a), (b), and (c). The patterns are shown in natural logarithm scale by photon counts. (h) Radial average over angle as a function of momentum transfer and spatial resolution. The diffraction intensity of Rice Dwarf Virus is much stronger than that of DNA-origami. To make the simulation reflect the experimental data, random detector noise, shot noise (Poisson noise), solid angle corrections, and polarization corrections were added to the simulated patterns. The calculation starts with reading the atom types and positions from the PDB file. After the theoretical intensity (in photon numbers) was obtained by the aforementioned equations, shot noise superimposed to the theoretical photon numbers.

With the help of


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detector’s gain under certain photon energy and geometry, crosstalk was taken into consideration. The ADUs per photon used is 130. The next step is the calculation of random noise. It was estimated based on the detector’s pixel RMS. Finally, the ADUs with noises were converted to the practical photon counts. Figure 6 shows 2D simulated results with noises. The main parameters used are the same as 3D simulations for soft and hard X-ray FELs (A summary list was provided in the Supporting Information Table S1). In 3D simulation section, the pulse energies are all fixed to 2 mJ. For 2D simulation with noises, serial pulse energies were employed. The 0.1 mJ pulse energy represents the BL3/BL2 of SACLA. A pulse energy of 0.8 mJ close to the current SPB/SFX conditions at the European-XFEL. The 2 mJ and 4 mJ designate the LCLS and LCLS-II CuRF, respectively. The use of diverse pulse energies makes simulation more relevant to the existing X-ray FEL facilities. Figure 6(a)-(d) shows diffraction patterns with a fixed orientation under hard X-ray regime (more patterns with random orientations are provided in the Supporting Information Figure S8 and S9). Related results for soft X-rays are shown in Figure 6(e)-(h). Obvious intensity difference can be identified from patterns. Detector defects are also apparent on the upper left side of each pattern. The total photon numbers per pattern of RDV experimental data is 4×106. However, for the strongest simulated patterns of DNA-origami with 4 mJ pulse energy, as shown in Figure 6(d) and 6(h), the total photon number are 1.4×105 and 7.2×104, respectively. For a more direct analysis, averaged radial curves are also outlined from the 8 simulated patterns. The intensity decreases fast approaching high-q direction. A pink reference line was fitted from the RDV experiment data, represents the non-hit backgrounds (Figure S10 and the details are provided in the Supporting Information). The peak around 0.68 nm-1 showed by the fitted background curve comes from the detector defects. From the two group curves shown in Figure


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6(i) and 6(j), we can estimate that the best theoretical full-period resolution for hard X-rays and soft X-rays used here are 11.4 nm and 31.4 nm. The reconstructed images (Figure S11 and descriptions are provided in the Supporting Information) demonstrated the same results. Due to the low photon counts and limited resolution, the inner structures were difficult to be identified.

Fig. 6. Simulated two-dimensional diffraction patterns combined with noises. (a)~(d) Twodimensional patterns of DNA-origami with a photon energy of 7.0 keV under serial pulse energies, 0.1 mJ, 0.8 mJ, 2.0 mJ, and 4.0 mJ. (e)~(h) Two-dimensional noisy patterns with 1.6 keV photon energy, and the pulse energies were the same as hard X-rays. (i), (j) Radial intensity distribution over momentum transfer and resolution for hard and soft X-ray FELs. A pink line


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was attached as a non-hit background reference. The fitted background data was generated from the published results. 25-28 The lessons learned from the simulations indicate that the imaging of DNA origami is challenging. Compared with the solid targets, the achievable signal is weak. It’s difficult to capture enough good single-hit data for phasing. Moreover, the sample was assumed in free states. Water rings from the liquid jet injectors or gas backgrounds from the aerosol injectors were omitted in this simulation. The conditions will be even worse if backgrounds from particle injectors were taken into account. Nevertheless, the lessons also enlighten us that signalenhanced methods and noise reduction methods development on software and X-ray instruments are of great significance. Attentions paid on the new apertures, high-efficiency X-ray mirrors, detectors, algorithms, and sample delivery are worth trying. CONCLUSION In this study, we conducted single-shot diffraction imaging of DNA-based structures at SACLA, Japan. The experiment was designed to capture the structure of DNA chains. However, only images of AuNPs were reconstructed. According to quantitative calculations of the coherent scattering cross-sections of gold and DNA, Mie scattering, and Porod’s law analysis, we found that the scattering ability of DNA is weak. The very thin DNA chains and weak scattering ability made them difficult to be detected under the current imaging resolution. In addition, the backgrounds from the fixed-target Si3N4 membrane and contaminants rapidly made the noise level equivalent to the sample signals with the increase in momentum transfer. To outline the necessary experimental conditions toward snapshots of DNA-based molecules, scaffolds, particles, and structures with X-ray lasers, numerical simulations with and without noise were implemented. The results indicate that it is challenging to determine the structures of DNA-


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origami scaffolds at high resolutions with current X-ray FEL facilities, such as LCLS and European-XFEL. Continuous efforts should be devoted to the sample delivery, X-ray instruments, experimental techniques, detectors, and data analysis. It’s meaningful to get to know the problems we are facing. There are still possibilities for capturing DNA-origami structures, which is necessary to overcome the experimental obstacles and advance the SPI technologies. Achieving the atomic resolution is truly a grand challenge. We believe that with the collection of more 2D frames in high signal levels by short X-ray laser pulses, 3D structures toward the high resolution could be eventually achieved. METHODS Sample synthesis. We employed super-origami as templates for anchoring 50-nm AuNPs on the prescribed docking sites. First, we constructed rhombus-shaped super-origami with 18 capture strands at specific positions. Then, the 50-nm AuNPs were anchored at specific sites on purified super-origami templates through DNA hybridization. The atomic force microscopy (AFM; Figure 3c) and scanning electron microscopy (SEM; Supporting Information Figure S1 c and d) images indicate that the 50-nm AuNPs were able to attach quantitatively and correctly to the super-origami templates. The assembling methods are as follows. Before use, the colloidal solutions of 50-nm AuNPs were functionalized with thiolated DNA according to the previously published method. 59, 60 The triangular DNA origami monomers were prepared according to Refs.

61, 62

The DNA-modified AuNP solution was added to the DNA

origami solution (purified) in a 0.6×TAE-Mg2+ buffer with a ratio of 2:1 for the 50-nm AuNP dimer. The mixtures were annealed from 45 to 15 °C at a rate of 0.1 °C/min to promote hybridization of the DNA on the AuNPs with the complementary capture strands on the DNA origami. The successful formation of the desired structures and separation from other products


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are accomplished by gel electrophoresis in a 0.5% agarose gel run at 100 V and are maintained at 4 °C by a surrounding ice bath. The desired bands were cut and DNA origami-templated AuNP clusters were extracted from the gel using the protocol given in Shih et al. 63 Sample preparation.

Nano- and bio-particles tend to aggregate when being dried. To

increase the number of single particles or structures, we ultra-sonicated the sample solution with target structures for 30~60s and repeated this several times. Before using the Si3N4 membrane, plasma cleaner was used to charge the membrane surface, to improve the wettability. In addition, the concentration and monodispersity of target structures were checked by the optical microscopy. Finally, the solution containing the isolated target structures was spin-coated on negatively charged Si3N4 membrane. ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website. Additional model and SEM images of the target DNA-based structure, pattern classification method details, consistency check with different phase retrieval algorithms, validation checks for final reconstructed results, representative patterns and images of DNA-based AuNPs, calculation details of golden-core and DNA-shell, representative 2D slices of 3D diffraction volumes, representative 2D patterns of DNA-origami with soft and hard X-rays, backgrounds from the simulated data and experiment data, reconstructions of simulated 2D noisy patterns were provided. In addition, simulation parameters were listed. (PDF) AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]


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Author Contributions H.J. directed the research; H.J. and C.F. designed the experiment; C.F., H. Liu synthesized and characterized the samples. Z.S., J.F., D.N., C.K., Y.K., J.P., S.K., C.S. and H.J. conducted the experiments; T.I., M.Y. and K.T. lead the design and constructions of X-ray FELs instruments. Z.S., H. Li conducted the numerical simulations. H. Li, Y.H. figured out the characterizations of the detector. Z.S., J.F. and H.J. analyzed the data and interpreted the results. Z.S. wrote the manuscript and all authors commented on the manuscript. The authors declare no conflict of interest. ACKNOWLEDGEMENT This work was supported by the National Natural Science Foundation of China (31430031, 21727817); the Major State Basic Research Development Program of China (2014CB910401, 2017YFA0504802); and the China Scholarship Council (201606220148). The experiments were performed at SACLA with the approval of JASRI and the program review committee. The simulations were performed at Linac Coherent Light Source, SLAC National Accelerator Laboratory, a national user facility operated by Stanford University for the U.S. Department of Energy, Office of Basic Energy Sciences (DE-AC02-76SF00515). REFERENCES (1) Emma, P.; Akre, R.; Arthur, J.; Bionta, R.; Bostedt, C.; Bozek, J.; Brachmann, A.; Bucksbaum, P.; Coffee, R.; Decker, F. J.; Ding, Y.; Dowell, D.; Edstrom, S.; Fisher, A.; Frisch, J.; Gilevich, S.; Hastings, J.; Hays, G.; Hering, P.; Huang, Z., et al., First Lasing and Operation of an Ångstrom-Wavelength Free-Electron Laser. Nat. Photonics 2010, 4, 641-647.


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(2) Ishikawa, T.; Aoyagi, H.; Asaka, T.; Asano, Y.; Azumi, N.; Bizen, T.; Ego, H.; Fukami, K.; Fukui, T.; Furukawa, Y.; Goto, S.; Hanaki, H.; Hara, T.; Hasegawa, T.; Hatsui, T.; Higashiya, A.; Hirono, T.; Hosoda, N.; Ishii, M.; Inagaki, T., et al., A Compact X-Ray Free-Electron Laser Emitting in the Sub-Angstrom Region. Nat. Photonics 2012, 6, 540-544. (3) Young, L.; Kanter, E. P.; Krässig, B.; Li, Y.; March, A. M.; Pratt, S. T.; Santra, R.; Southworth, S. H.; Rohringer, N.; DiMauro, L. F.; Doumy, G.; Roedig, C. A.; Berrah, N.; Fang, L.; Hoener, M.; Bucksbaum, P. H.; Cryan, J. P.; Ghimire, S.; Glownia, J. M.; Reis, D. A., et al., Femtosecond Electronic Response of Atoms to Ultra-Intense X-Rays. Nature 2010, 466, 56-61. (4) Seibert, M. M.; Ekeberg, T.; Maia, F. R. N. C.; Svenda, M.; Andreasson, J.; Jonsson, O.; Odic, D.; Iwan, B.; Rocker, A.; Westphal, D.; Hantke, M.; DePonte, D. P.; Barty, A.; Schulz, J.; Gumprecht, L.; Coppola, N.; Aquila, A.; Liang, M.; White, T. A.; Martin, A., et al., Single Mimivirus Particles Intercepted and Imaged with an X-Ray Laser. Nature 2011, 470, 78-81. (5) Boutet, S.; Lomb, L.; Williams, G. J.; Barends, T. R. M.; Aquila, A.; Doak, R. B.; Weierstall, U.; DePonte, D. P.; Steinbrener, J.; Shoeman, R. L.; Messerschmidt, M.; Barty, A.; White, T. A.; Kassemeyer, S.; Kirian, R. A.; Seibert, M. M.; Montanez, P. A.; Kenney, C.; Herbst, R.; Hart, P., et al., High-Resolution Protein Structure Determination by Serial Femtosecond Crystallography. Science 2012, 337, 362-364. (6) Chapman, H. N.; Fromme, P.; Barty, A.; White, T. A.; Kirian, R. A.; Aquila, A.; Hunter, M. S.; Schulz, J.; DePonte, D. P.; Weierstall, U.; Doak, R. B.; Maia, F. R. N. C.; Martin, A. V.; Schlichting, I.; Lomb, L.; Coppola, N.; Shoeman, R. L.; Epp, S. W.; Hartmann, R.; Rolles, D., et al., Femtosecond X-Ray Protein Nanocrystallography. Nature 2011, 470, 73-77. (7) Vinko, S. M.; Ciricosta, O.; Cho, B. I.; Engelhorn, K.; Chung, H. K.; Brown, C. R. D.; Burian, T.; Chalupsky, J.; Falcone, R. W.; Graves, C.; Hajkova, V.; Higginbotham, A.; Juha, L.;


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Krzywinski, J.; Lee, H. J.; Messerschmidt, M.; Murphy, C. D.; Ping, Y.; Scherz, A.; Schlotter, W., et al., Creation and Diagnosis of a Solid-Density Plasma with an X-Ray Free-Electron Laser. Nature 2012, 482, 59-62. (8) Garman, E. F.; Weik, M., Radiation Damage to Biological Macromolecules: Some Answers and More Questions. J. Synchrotron Radiat. 2013, 20, 1-6. (9) Neutze, R.; Wouts, R.; Spoel, D.; Weckert, E.; Hajdu, J., Potential for Biomolecular Imaging with Femtosecond X-Ray Pulses. Nature 2000, 406, 752-757. (10) Chapman, H. N.; Barty, A.; Bogan, M. J.; Boutet, S.; Frank, M.; Hau-Riege, S. P.; Marchesini, S.; Woods, B. W.; Bajt, S.; Benner, W. H.; London, R. A.; Plonjes, E.; Kuhlmann, M.; Treusch, R.; Dusterer, S.; Tschentscher, T.; Schneider, J. R.; Spiller, E.; Moller, T.; Bostedt, C., et al., Femtosecond Diffractive Imaging with a Soft-X-Ray Free-Electron Laser. Nat. Phys. 2006, 2, 839-843. (11) Kimura, T.; Joti, Y.; Shibuya, A.; Song, C.; Kim, S.; Tono, K.; Yabashi, M.; Tamakoshi, M.; Moriya, T.; Oshima, T.; Ishikawa, T.; Bessho, Y.; Nishino, Y., Imaging Live Cell in MicroLiquid Enclosure by X-Ray Laser Diffraction. Nat. Commun. 2014, 5, 3052. (12) Schot, G. v. d.; Svenda, M.; Maia, F. R. N. C.; Hantke, M.; DePonte, D. P.; Seibert, M. M.; Aquila, A.; Schulz, J.; Kirian, R.; Liang, M.; Stellato, F.; Iwan, B.; Andreasson, J.; Timneanu, N.; Westphal, D.; Almeida, F. N.; Odic, D.; Hasse, D.; Carlsson, G. H.; Larsson, D. S. D., et al., Imaging Single Cells in a Beam of Live Cyanobacteria with an X-Ray Laser. Nat. Commun. 2015, 6, 5704. (13) Ekeberg, T.; Svenda, M.; Abergel, C.; Maia, F. R. N. C.; Seltzer, V.; Claverie, J.-M.; Hantke, M.; Jönsson, O.; Nettelblad, C.; Van Der Schot, G., Three-Dimensional Reconstruction


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Reis, D. A.; Robinson, I. K., Imaging Transient Melting of a Nanocrystal Using an X-Ray Laser. Proc. Natl. Acad. Sci. U. S. A. 2015, 112, 7444-7448. (19) Clark, J. N.; Beitra, L.; Xiong, G.; Higginbotham, A.; Fritz, D. M.; Lemke, H. T.; Zhu, D.; Chollet, M.; Williams, G. J.; Messerschmidt, M.; Abbey, B.; Harder, R. J.; Korsunsky, A. M.; Wark, J. S.; Robinson, I. K., Ultrafast Three-Dimensional Imaging of Lattice Dynamics in Individual Gold Nanocrystals. Science 2013, 341, 56-59. (20) Bogan, M. J.; Benner, W. H.; Boutet, S.; Rohner, U.; Frank, M.; Barty, A.; Seibert, M. M.; Maia, F.; Marchesini, S.; Bajt, S.; Woods, B.; Riot, V.; Hau-Riege, S. P.; Svenda, M.; Marklund, E.; Spiller, E.; Hajdu, J.; Chapman, H. N., Single Particle X-Ray Diffractive Imaging. Nano Lett. 2008, 8, 310-316. (21) Loh, N. D.; Hampton, C. Y.; Martin, A. V.; Starodub, D.; Sierra, R. G.; Barty, A.; Aquila, A.; Schulz, J.; Lomb, L.; Steinbrener, J.; Shoeman, R. L.; Kassemeyer, S.; Bostedt, C.; Bozek, J.; Epp, S. W.; Erk, B.; Hartmann, R.; Rolles, D.; Rudenko, A.; Rudek, B., et al., Fractal Morphology, Imaging and Mass Spectrometry of Single Aerosol Particles in Flight. Nature 2012, 486, 513-517. (22) Aquila, A.; Barty, A.; Bostedt, C.; Boutet, S.; Carini, G.; dePonte, D.; Drell, P.; Doniach, S.; Downing, K. H.; Earnest, T.; Elmlund, H.; Elser, V.; Gühr, M.; Hajdu, J.; Hastings, J.; HauRiege, S. P.; Huang, Z.; Lattman, E. E.; Maia, F. R. N. C.; Marchesini, S., et al., The Linac Coherent Light Source Single Particle Imaging Road Map. Struct. Dyn. 2015, 2, 041701. (23) Daurer, B. J.; Okamoto, K.; Bielecki, J.; Maia, F. R. N. C.; Muhlig, K.; Seibert, M. M.; Hantke, M. F.; Nettelblad, C.; Benner, W. H.; Svenda, M.; Timneanu, N.; Ekeberg, T.; Loh, N. D.; Pietrini, A.; Zani, A.; Rath, A. D.; Westphal, D.; Kirian, R. A.; Awel, S.; Wiedorn, M. O., et


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al., Experimental Strategies for Imaging Bioparticles with Femtosecond Hard X-Ray Pulses. IUCrJ 2017, 4, 251-262. (24) Donatelli, J. J.; Sethian, J. A.; Zwart, P. H., Reconstruction from Limited Single-Particle Diffraction Data Via Simultaneous Determination of State, Orientation, Intensity, and Phase. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 7222-7227. (25) Munke, A.; Andreasson, J.; Aquila, A.; Awel, S.; Ayyer, K.; Barty, A.; Bean, R. J.; Berntsen, P.; Bielecki, J.; Boutet, S.; Bucher, M.; Chapman, H. N.; Daurer, B. J.; DeMirci, H.; Elser, V.; Fromme, P.; Hajdu, J.; Hantke, M. F.; Higashiura, A.; Hogue, B. G., et al., Coherent Diffraction of Single Rice Dwarf Virus Particles Using Hard X-Rays at the Linac Coherent Light Source. Sci. Data 2016, 3, 160064. (26) Reddy, H. K. N.; Yoon, C. H.; Aquila, A.; Awel, S.; Ayyer, K.; Barty, A.; Berntsen, P.; Bielecki, J.; Bobkov, S.; Bucher, M.; Carini, G. A.; Carron, S.; Chapman, H.; Daurer, B.; DeMirci, H.; Ekeberg, T.; Fromme, P.; Hajdu, J.; Hanke, M. F.; Hart, P., et al., Coherent Soft XRay Diffraction Imaging of Coliphage PR772 at the Linac Coherent Light Source. Sci. Data 2017, 4, 170079. (27) Hosseinizadeh, A.; Mashayekhi, G.; Copperman, J.; Schwander, P.; Dashti, A.; Sepehr, R.; Fung, R.; Schmidt, M.; Yoon, C. H.; Hogue, B. G.; Williams, G. J.; Aquila, A.; Ourmazd, A., Conformational Landscape of a Virus by Single-Particle X-Ray Scattering. Nat. Methods 2017, 14, 877-881. (28) Kurta, R. P.; Donatelli, J. J.; Yoon, C. H.; Berntsen, P.; Bielecki, J.; Daurer, B. J.; DeMirci, H.; Fromme, P.; Hantke, M. F.; Maia, F. R. N. C.; Munke, A.; Nettelblad, C.; Pande, K.; Reddy, H. K. N.; Sellberg, J. A.; Sierra, R. G.; Svenda, M.; van der Schot, G.; Vartanyants, I. A.;


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