Coherent Diffractive Imaging of Single Particles

Our group develops novel methods to achieve the foundational goals of single-particle diffractive imaging using XFEL pulses.

Single-particle diffractive imaging is one of the key foundational goals behind the establishment of X-ray free-electron laser (XFELs) facilities and is envisioned to revolutionize the way single-molecules are imaged. Single-molecule diffractive imaging at ultrafast time-scales of atomic and electronic motions is expected to provide a wealth of information to map the complete conformational landscape of molecules both at ground and excited states, which otherwise can not be obtained through conventional molecular imaging methods based on the signal-averaging of ensemble as in crystallography [1].

Highly intense femtosecond XFEL pulses outrun radiation damage and open up the possibility of radiation-damage-free high-resolution imaging of nanoparticles, macromolecules, and viruses at room temperature. Though XFELs impart large doses onto matter in a single-pulse, the short femtosecond pulse duration plays the key role in outrunning radiation damage effects [2]. The extreme intensity of XFELs—billion times brighter than the synchrotron light—providing enough photons per pulse and the coherence of the X-ray laser together enable the imaging of uncrystallized aperiodic single-particles obviating the need for crystals—especially it frees from the herculean task of crystallizing difficult-to-crystallize macromoleucules. The coherence of the incident XFEL pulse leads to a diffraction pattern or two-dimensional Fourier transform snapshot of the target single-particles in the far-field in the reciprocal space, which is recorded on a detector and computationally processed to solve the electron density map of the target structure (see Figure 1). Compared to the real-space imaging with lenses, diffractive imaging is lens-aberration-free, the resolution is diffraction and the radiation dose limited, and is deemed suitable for investigating irreversible or non-cyclic processes at femto- or attosecond timescales [3].

The XFEL pulses incident on the target particles is short enough to carry the information or the diffraction pattern of the target object in the scattered photons before the particle is destroyed due to the onset of radiation damage processes such as ionization, atomic positional rearrangements and Coulomb explosion, and this “diffraction-before-destruction” records the pristine radiation-damage-free state of the target while the atomic motions are frozen in the femtosecond timescale. Since only one two-dimensional snapshot can be taken per pulse, a continuous stream of reproducible particles needs to be supplied in a serial-fashion to take enough noisy 2D single-shots for the reconstruction of a complete 3D structure (see Figure 1) [3]. The number of required shots depends on the size of the target, the signal-to-noise ratio, and the desired resolution. Our group helped demonstrate the principle of ‘diffraction-before-destruction’ using the world's first free-electron laser FLASH in 2006 and showed that coherent diffractive imaging (CDI) of objects can be successfully performed with femtosecond FEL pulses, albeit in this case two-dimensional lithographic objects were imaged [4]. Subsequently, our group demonstrated femtosecond time-delay X-ray holography [5] and nanoscale ultrafast dynamics of exploding 2D objects at FLASH [6]. Also the group helped demonstrate the first aerosol single-particle diffractive imaging of free-flying nanoscale objects with FELs at FLASH in 2007 by imaging nanoparticles of sucrose-DNA mixture that were electrospray injected, aerosolized and delivered to the FEL beam using an aerodynamic lens stack [7].

Since the first proof-of-principle experiments, our group has led or been part of several seminal single-particle imaging (SPI) experiments at XFEL facilities together with our collaborators, for example in imaging the giant viruses [8, 9] and the aerosol soot nanoparticles [10]. We also contribute to the community-led SPI initiatives at the Linac Coherent Light Source (LCLS), Stanford, USA [11] and the European XFEL (EuXFEL), Hamburg, Germany, while we still continue to execute our own SPI experiments.

Figure 1. XFEL single-particle diffractive imaging pipeline involves recording single-shot diffraction patterns of the target-object before the destruction due to ionization and Coloumb explosion (top), computational purification for expected single-particle diffraction patterns, orientation determination, classification into 2D class averages, and the 3D merging of intensities in the reciprocal space—after this step, an inverse Fourier transform of the intensities alone is not enough and one needs to determine the missing phase information to solve the electron density map of the structure—this is achieved using iterative phase retrieval algorithms (bottom). Note: the lensless imaging method also can be used with coherent electrons to reconstruct the Coulomb potential map of the target sample. Image source: Henry Chapman, CFEL. Science, 2007, 316, 1444-48.


The challenge: Since single-macromolecules scatter very weakly, achieving high-resolution 3D reconstruction of a macromolecule has been a longstanding challenge, and requires developments in the direction of sample delivery methods—friendly to native or physiological states of macromolecules—providing high hit-rate while achieving sufficiently low-background scattering to record high-resolution signal above the background noise, novel x-ray optics and sensitive detectors with higher dynamic range than the existing ones. Meanwhile, in the voyage of imaging single-molecules, our group demonstrated the benefit of XFELs by developing the popular serial femtosecond crystallography method using tiny crystals to image biologically important macromolecules, enzyme kinetics and light-induced ultrafast dynamics at room temperature.

The group has the all necessary infrastructure, expertise and focuses on all aspects required for high-resolution single-particle imaging such as simulations, instrumentation, x-ray optics, holographic methods, sample preparation and characterization, low-background sample delivery methods, online and offline data-analysis, image classification and reconstruction algorithms, and softwares to achieve the final single-molecule structure. In the context of XFEL-SPI, currently we are interested in the structure determination of inorganic nanoparticles, biological macromolecules, fibers, viruses, and artificial nucleic acid nanostructures. As an innovative approach, the group also focuses on amplifying weak single-molecule signal through arbitrary alignment of macromolecules and deploying strongly scattering holographic references to enhance the signal of weakly diffracting single-molecules [12].

The current state-of-the-art three-dimensional reconstruction of a bio-particle is around 9-nm resolution in case of a large virus (c.a. 2020 CE) [13]. Achieving high-resolution single-macromolecule structure determination with x-ray lasers is a grand challenge and requires multidisciplinary efforts and developments [14,15]. With the advent of high-repetition rate MHz XFELs, improved x-ray optics, holographic methods, innovative sample-preparation and delivery methods unique to XFEL imaging conditions, and efficient algorithms, soon we are set to enter the domain of sub-nm resolution, million-pattern XFEL imaging of single-particles.


Structural DNA nanotechnology offers the possibility of creating almost any desired nanoscale DNA-origami shapes, which can be model samples for XFEL imaging and also customizable devices/platforms to carry out single-molecule imaging studies. Programmable nature of DNA enables the organization of non-nucleic acid materials, such as nanoparticles, viruses, and proteins at spatially addressable locations, in complex three-dimensional geometries, with sub-nm scale precision and likely helps in molecular scaffold or holographic reference based imaging of weakly diffracting single-molecules at high-resolution.


Human Frontier Science Program (RGP0010/2017), CUI: Advanced Imaging of the Matter, DESY-Helmholtz Association


K.J. Gaffney, H.N. Chapman, Imaging Atomic Structure and Dynamics with Ultrafast X-ray Scattering, Science 316(5830) (2007) 1444.