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Gold nanoparticles (AuNPs) are increasingly used in applications across the biomedical domain, yet their long-term biodistribution and biocompatibility remain poorly understood. Conventional brightfield microscopy imaging techniques often fail to detect AuNPs due to optical diffraction limits and lack of chromogenic contrast. Understanding the biodistribution and ultimate fate of these nonbiodegradable NPs is crucial for further development of AuNP-based therapeutics and diagnostics. Here, we present a label-free multiphoton luminescence (MPL) imaging workflow that enables sensitive detection of AuNPs in liver histology sections, even 1 year after intravenous (IV) administration. MPL imaging exploits the intrinsic nonlinear optical properties of AuNPs to generate broadband emission under ultrafast pulsed laser excitation, enabling subcellular localization without exogenous labels while having the ability to rapidly image entire organ sections. The intrinsic, distinct broadband MPL emission produced by gold allows us to study these NPs in their biological context without extrinsic labels while also faithfully representing the surrounding tissue architecture via autofluorescence and second harmonic generation. We demonstrate that MPL imaging detects up to 98% more AuNP-positive regions than brightfield microscopy in challenging low-dose (1 nM) conditions and requires no modification of standard histology workflows. Correlative imaging with SEM–EDS confirms high spatial specificity (AUC = 0.955) of MPL for AuNP localization. Dose-dependent retention patterns were observed across liver tissue, and MPL analysis showed strong correlation with ICP–MS quantification. Importantly, histological and immunohistochemical analyses (Masson’s trichrome, CD3, and TUNEL) revealed no significant fibrosis, immune activation, or apoptosis in liver tissue at either low (1 nM) or high (10 nM) doses at 1 year post IV administration. These findings establish MPL imaging as a robust, label-free tool for long-term tracking of AuNPs in biological tissue and highlight its potential for improving biodistribution and safety assessments. 

An experimental platform for dynamic diamond anvil cell (dDAC) research has been developed at the High Energy Density (HED) Instrument at the European X-ray Free Electron Laser (European XFEL). Advantage was taken of the high repetition rate of the European XFEL (up to 4.5 MHz) to collect pulse-resolved MHz X-ray diffraction data from samples as they are dynamically compressed at intermediate strain rates (≤10³ s⁻¹), where up to 352 diffraction images can be collected from a single pulse train. The set-up employs piezo-driven dDACs capable of compressing samples in ≥340 µs, compatible with the maximum length of the pulse train (550 µs). Results from rapid compression experiments on a wide range of sample systems with different X-ray scattering powers are presented. A maximum compression rate of 87 TPa s⁻¹was observed during the fast compression of Au, while a strain rate of ∼1100 s⁻¹was achieved during the rapid compression of N₂at 23 TPa s⁻¹. 

The European X-ray Free-Electron Laser (FEL) became the first operational high-repetition-rate hard X-ray FEL with first lasing in May 2017. Biological structure determination has already benefitted from the unique properties and capabilities of X-ray FELs, predominantly through the development and application of serial crystallography. The possibility of now performing such experiments at data rates more than an order of magnitude greater than previous X-ray FELs enables not only a higher rate of discovery but also new classes of experiments previously not feasible at lower data rates. One example is time-resolved experiments requiring a higher number of time steps for interpretation, or structure determination from samples with low hit rates in conventional X-ray FEL serial crystallography. Following first lasing at the European XFEL, initial commissioning and operation occurred at two scientific instruments, one of which is the Single Particles, Clusters and Biomolecules and Serial Femtosecond Crystallography (SPB/SFX) instrument. This instrument provides a photon energy range, focal spot sizes and diagnostic tools necessary for structure determination of biological specimens. The instrumentation explicitly addresses serial crystallography and the developing single particle imaging method as well as other forward-scattering and diffraction techniques. This paper describes the major science cases of SPB/SFX and its initial instrumentation – in particular its optical systems, available sample delivery methods, 2D detectors, supporting optical laser systems and key diagnostic components. The present capabilities of the instrument will be reviewed and a brief outlook of its future capabilities is also described. 

Abstract The emergence of high repetition-rate X-ray free-electron lasers (XFELs) powered by superconducting accelerator technology enables the measurement of significantly more experimental data per day than was previously possible. The European XFEL is expected to provide 27,000 pulses per second, over two orders of magnitude more than any other XFEL. The increased pulse rate is a key enabling factor for single-particle X-ray diffractive imaging, which relies on averaging the weak diffraction signal from single biological particles. Taking full advantage of this new capability requires that all experimental steps, from sample preparation and delivery to the acquisition of diffraction patterns, are compatible with the increased pulse repetition rate. Here, we show that single-particle imaging can be performed using X-ray pulses at megahertz repetition rates. The results obtained pave the way towards exploiting high repetition-rate X-ray free-electron lasers for single-particle imaging at their full repetition rate. 

Abstract Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) allows structure determination of membrane proteins and time-resolved crystallography. Common liquid sample delivery continuously jets the protein crystal suspension into the path of the XFEL, wasting a vast amount of sample due to the pulsed nature of all current XFEL sources. The European XFEL (EuXFEL) delivers femtosecond (fs) X-ray pulses in trains spaced 100 ms apart whereas pulses within trains are currently separated by 889 ns. Therefore, continuous sample delivery via fast jets wastes >99% of sample. Here, we introduce a microfluidic device delivering crystal laden droplets segmented with an immiscible oil reducing sample waste and demonstrate droplet injection at the EuXFEL compatible with high pressure liquid delivery of an SFX experiment. While achieving ~60% reduction in sample waste, we determine the structure of the enzyme 3-deoxy-D-manno-octulosonate-8-phosphate synthase from microcrystals delivered in droplets revealing distinct structural features not previously reported.