The advent of ultrafast X-ray free-electron lasers (XFELs) opens the tantalizing possibility of the atomic-resolution imaging of reproducible objects such as viruses, nanoparticles, single molecules, clusters, and perhaps biological cells, achieving a resolution for single particle imaging better than a few tens of nanometers. Improving upon this is a significant challenge which has been the focus of a global single particle imaging (SPI) initiative launched in December 2014 at the Linac Coherent Light Source (LCLS), SLAC National Accelerator Laboratory, USA. A roadmap was outlined, and significant multi-disciplinary effort has since been devoted to work on the technical challenges of SPI such as radiation damage, beam characterization, beamline instrumentation and optics, sample preparation and delivery and algorithm development at multiple institutions involved in the SPI initiative. Currently, the SPI initiative has achieved 3D imaging of rice dwarf virus (RDV) and coliphage PR772 viruses at ~10 nm resolution by using soft X-ray FEL pulses at the Atomic Molecular and Optical (AMO) instrument of LCLS. Meanwhile, diffraction patterns with signal above noise up to the corner of the detector with a resolution of ~6 Ångström (Å) were also recorded with hard X-rays at the Coherent X-ray Imaging (CXI) instrument, also at LCLS. Achieving atomic resolution is truly a grand challenge and there is still a long way to go in light of recent developments in electron microscopy. However, the potential for studying dynamics at physiological conditions and capturing ultrafast biological, chemical and physical processes represents a tremendous potential application, attracting continued interest in pursuing further method development. In this paper, we give a brief introduction of SPI developments and look ahead to further method development.

Knowledge of the interactions between nanomaterials and large-size mammalian cells, including cellular uptake, intracellular localization and translocation, has greatly advanced nanomedicine and nanotoxicology. Imaging techniques that can locate nanomaterials within the structures of intact large-size cells at nanoscale resolution play crucial roles in acquiring this knowledge. Here, the quantitative imaging of intracellular nanomaterials in three dimensions was performed by combining dual-energy contrast X-ray microscopy and an iterative tomographic algorithm termed equally sloped tomography (EST). Macrophages with a size of ∼20 µm that had been exposed to the potential antitumour agent [Gd@C₈₂(OH)₂₂]_nwere investigated. Large numbers of nanoparticles (NPs) aggregated within the cell and were mainly located in phagosomes. No NPs were observed in the nucleus. Imaging of the nanomedicine within whole cells advanced the understanding of the high-efficiency antitumour activity and the low toxicity of this agent. This imaging technique can be used to probe nanomaterials within intact large-size cells at nanometre resolution uniformly in three dimensions and may greatly benefit the fields of nanomedicine and nanotoxicology.

Characterizing relationships between Zn ²⁺ , insulin, and insulin vesicles is of vital importance to the study of pancreatic beta cells. However, the precise content of Zn ²⁺ and the specific location of insulin inside insulin vesicles are not clear, which hinders a thorough understanding of the insulin secretion process and diseases caused by blood sugar dysregulation. Here, we demonstrated the colocalization of Zn ²⁺ and insulin in both single extracellular insulin vesicles and pancreatic beta cells by using an X-ray scanning coherent diffraction imaging (ptychography) technique. We also analyzed the elemental Zn ²⁺ and Ca ²⁺ contents of insulin vesicles using electron microscopy and energy dispersive spectroscopy (EDS) mapping. We found that the presence of Zn ²⁺ is an important characteristic that can be used to distinguish insulin vesicles from other types of vesicles in pancreatic beta cells and that the content of Zn ²⁺ is proportional to the size of insulin vesicles. By using dual-energy contrast X-ray microscopy and scanning transmission X-ray microscopy (STXM) image stacks, we observed that insulin accumulates in the off-center position of extracellular insulin vesicles. Furthermore, the spatial distribution of insulin vesicles and their colocalization with other organelles inside pancreatic beta cells were demonstrated using three-dimensional (3D) imaging by combining X-ray ptychography and an equally sloped tomography (EST) algorithm. This study describes a powerful method to univocally describe the location and quantitative analysis of intracellular insulin, which will be of great significance to the study of diabetes and other blood sugar diseases.

Characterizing interactions between microbial cells and their specific inhibitory drugs is essential for developing effective drugs and understanding the therapeutic mechanism. Functional metal nanoclusters can be effective inhibitory agents against microorganisms according to various characterization methods, but quantitative three-dimensional (3D) spatial structural analysis of intact cells is lacking. Herein, using coherent X-ray diffraction imaging, we performed in situ 3D visualization of unstained Staphylococcus aureus cells treated with peptide-mineralized Au-cluster probes at a resolution of ∼47 nm. Subsequent 3D mass-density mapping and quantitative structural analyses of S. aureus in different degrees of destruction showed that the bacterial cell wall was damaged and cytoplasmic constituents were released from cells, confirming the significant antibacterial effects of the Au-cluster probe. This study provides a promising nondestructive approach for quantitative imaging and paves the way for further research into microbe-inhibitor drug interactions.