An Overview of Microcrystal Electron Diffraction (MicroED).

The bedrock of drug discovery and a key tool for understanding cellular function and drug mechanisms of action is the structure determination of chemical compounds, peptides, and proteins. The development of new structure characterization tools, particularly those that fill critical gaps in existing methods, presents important steps forward for structural biology and drug discovery. The emergence of microcrystal electron diffraction (MicroED) expands the application of cryo-electron microscopy to include samples ranging from small molecules and membrane proteins to even large protein complexes using crystals that are one-billionth the size of those required for X-ray crystallography. This review outlines the conception, achievements, and exciting future trajectories for MicroED, an important addition to the existing biophysical toolkit.

Biodegradable Long-Acting Injectables: Platform Technology and Industrial Challenges.

Long-acting injectables have been used to benefit patients with chronic diseases. So far, several biodegradable long-acting platform technologies including drug-loaded polymeric microparticles, implants (preformed and in situ forming), oil-based solutions, and aqueous suspension have been established. In this chapter, we summarize all the marketed technology platforms and discuss their challenges regarding development including but not limited to controlling drug release, particle size, stability, sterilization, scale-up manufacturing, etc. Finally, we discuss important criteria to consider for the successful development of long-acting injectables.

14N NMR of magnetically oriented microcrystals.

14N NMR of magnetically oriented microcrystals is reported. With a home-built 1H-13C-14N probe capable of modulating the rotation of the sample around the axis normal to the magnetic field, magnetically oriented microcrystal suspension (MOMS) of l-alanine is made. 14N NMR spectra acquired with various timings during intermittent rotation lead to a rotation pattern of the MOMS similar to that of a single crystal. The effect of orientational distribution of the microcrystals to broadening of the resonance line is discussed.

Rapid Structure Determination of Ranitidine Hydrochloride API in Two Crystal Forms Using Microcrystal Electron Diffraction.

The solid-state properties of drug candidates play a crucial role in their selection. Quality control of active pharmaceutical ingredients (APIs) based on their structural information involves ensuring a consistent crystal form and controlling water and residual solvent contents. However, traditional crystallographic techniques have limitations and require high-quality single crystals for structural analysis. Microcrystal electron diffraction (microED) overcomes these challenges by analyzing difficult-to-crystallize or small-quantity samples, making it valuable for efficient drug development. In this study, microED analysis was able to rapidly determine the configuration of two crystal forms (Forms 1, 2) of the API ranitidine hydrochloride. The structures obtained with microED are consistent with previous structures determined by X-ray diffraction, indicating microED is a useful tool for rapidly analyzing molecular structures in drug development and materials science research.

Compact and Fully Integrated LED Quantum Sensor Based on NV Centers in Diamond.

Quantum magnetometry based on optically detected magnetic resonance (ODMR) of nitrogen vacancy centers in diamond nano or microcrystals is a promising technology for sensitive, integrated magnetic-field sensors. Currently, this technology is still cost-intensive and mainly found in research. Here we propose one of the smallest fully integrated quantum sensors to date based on nitrogen vacancy (NV) centers in diamond microcrystals. It is an extremely cost-effective device that integrates a pump light source, photodiode, microwave antenna, filtering and fluorescence detection. Thus, the sensor offers an all-electric interface without the need to adjust or connect optical components. A sensitivity of 28.32nT/Hz and a theoretical shot noise limited sensitivity of 2.87 nT/Hz is reached. Since only generally available parts were used, the sensor can be easily produced in a small series. The form factor of (6.9 × 3.9 × 15.9) mm3 combined with the integration level is the smallest fully integrated NV-based sensor proposed so far. With a power consumption of around 0.1W, this sensor becomes interesting for a wide range of stationary and handheld systems. This development paves the way for the wide usage of quantum magnetometers in non-laboratory environments and technical applications.

Circularly Polarized Lasing from Helical Superstructures of Chiral Organic Molecules.

Chiral supramolecular aggregates have the potential to explore circularly polarized lasing with large dissymmetry factors. However, the controllable assembly of chiral superstructures towards deterministic circularly polarized laser emission remains elusive. Here, we design a pair of chiral organic molecules capable of stacking into a pair of definite helical superstructures in microcrystals, which enables circularly polarized lasing with deterministic chirality and high dissymmetry factors. The microcrystals function as optical cavities and gain media simultaneously for laser oscillations, while the supramolecular helices endow the laser emission with strong and opposite chirality. As a result, the microcrystals of two enantiomers allow for circularly polarized laser emission with opposite chirality and high dissymmetry factors up to ~1.0. This work demonstrates the chiral supramolecular assemblies as an excellent platform for high-performance circularly polarized lasers.

Intense Circularly Polarized Luminescence Induced by Chiral Supramolecular Assembly: The Importance of Intermolecular Electronic Coupling.

Herein we report on circularly polarized luminescence (CPL) emission originating from supramolecular chirality of organic microcrystals with a |glum| value up to 0.11. The microcrystals were prepared from highly emissive difluoroboron ß-diketonate (BF2dbk) dyes R-1 or S-1 with chiral binaphthol (BINOL) skeletons. R-1 and S-1 exhibit undetectable CPL signals in solution but manifest intense CPL emission in their chiral microcrystals. The chiral superstructures induced by BINOL skeletons were confirmed by XRD analysis. Spectral analysis and theoretical calculations indicate that intermolecular electronic coupling, mediated by the asymmetric stacking in the chiral superstructures, effectively alters excited-state electronic structures and facilitates electron transitions perpendicular to BF2bdk planes. The coupling increases cosθµ,m from 0.05 (monomer) to 0.86 (tetramer) and triggers intense optical activity of BF2bdk. The results demonstrate that optical activity of chromophores within assemblies can be regulated by both orientation and extent of intermolecular electronic couplings.

Adaptive Helical Chirality in Supramolecular Microcrystals for Circularly Polarized Lasing.

Chiral organic molecules offer a promising platform for exploring circularly polarized lasing, which, however, faces a great challenge that the spatial separation of molecular chiral and luminescent centers limits chiroptical activity. Here we develop a helically chiral supramolecular system with completely overlapped chiral and luminescent units for realizing high-performance circularly polarized lasing. Adaptive helical chirality is obtained by incorporating chiral agents into organic microcrystals. Benefiting from the efficient coupling of stimulated emission with the adaptive helical chirality, the supramolecular microcrystals enable high-performance circularly polarized lasing emission with dissymmetry factors up to ~0.7. This work opens up the way to rational design of chiral organic materials for circularly polarized lasing.

MicroED structure of lipid-embedded mammalian mitochondrial voltage-dependent anion channel.

A structure of the murine voltage-dependent anion channel (VDAC) was determined by microcrystal electron diffraction (MicroED). Microcrystals of an essential mutant of VDAC grew in a viscous bicelle suspension, making it unsuitable for conventional X-ray crystallography. Thin, plate-like crystals were identified using scanning-electron microscopy (SEM). Crystals were milled into thin lamellae using a focused-ion beam (FIB). MicroED data were collected from three crystal lamellae and merged for completeness. The refined structure revealed unmodeled densities between protein monomers, indicative of lipids that likely mediate contacts between the proteins in the crystal. This body of work demonstrates the effectiveness of milling membrane protein microcrystals grown in viscous media using a focused ion beam for subsequent structure determination by MicroED. This approach is well suited for samples that are intractable by X-ray crystallography. To our knowledge, the presented structure is a previously undescribed mutant of the membrane protein VDAC, crystallized in a lipid bicelle matrix and solved by MicroED.

On the Microcrystal Structure of Sputtered Cu Films Deposited on Si(100) Surfaces: Experiment and Integrated Multiscale Simulation.

Sputtered Cu/Si thin films were experimentally prepared at different sputtering pressures and characterized using X-ray diffraction (XRD) and an atomic force microscope (AFM). Simultaneously, an application-oriented simulation approach for magnetron sputtering deposition was proposed in this work. In this integrated multiscale simulation, the sputtered atom transport was modeled using the Monte Carlo (MC) and molecular dynamics (MD) coupling method, and the deposition of sputtered atoms was simulated using the MD method. This application-oriented simulation approach was used to simulate the growth of Cu/Si(100) thin films at different sputtering pressures. The experimental results unveiled that, as the sputtering pressure decreased from 2 to 0.15 Pa, the surface roughness of Cu thin films gradually decreased; (111)-oriented grains were dominant in Cu thin films and the crystal quality of the Cu thin film was gradually improved. The simulation results were consistent with the experimental characterization results. The simulation results revealed that the transformation of the film growth mode from the Volmer-Weber growth mode to the two-dimensional layered growth mode resulted in a decrease in the surface roughness of Cu thin films; the increase in the amorphous compound CuSix and the hcp copper silicide with the decrease in the sputtering pressure was responsible for the improvement of the crystal quality of the Cu thin film. This work proposed a more realistic, integrated simulation scheme for magnetron sputtering deposition, providing theoretical guidance for the efficient preparation of high-quality sputtered films.

Serial macromolecular crystallography at ALBA Synchrotron Light Source.

The increase in successful adaptations of serial crystallography at synchrotron radiation sources continues. To date, the number of serial synchrotron crystallography (SSX) experiments has grown exponentially, with over 40 experiments reported so far. In this work, we report the first SSX experiments with viscous jets conducted at ALBA beamline BL13-XALOC. Small crystals (15-30 µm) of five soluble proteins (lysozyme, proteinase K, phycocyanin, insulin and α-spectrin-SH3 domain) were suspended in lipidic cubic phase (LCP) and delivered to the X-ray beam with a high-viscosity injector developed at Arizona State University. Complete data sets were collected from all proteins and their high-resolution structures determined. The high quality of the diffraction data collected from all five samples, and the lack of specific radiation damage in the structures obtained in this study, confirm that the current capabilities at the beamline enables atomic resolution determination of protein structures from microcrystals as small as 15 µm using viscous jets at room temperature. Thus, BL13-XALOC can provide a feasible alternative to X-ray free-electron lasers when determining snapshots of macromolecular structures.

Serial macromolecular crystallography at ALBA Synchrotron Light Source. Erratum.

A revised version of Table 2 of Martin-Garcia et al. [J. Synchrotron Rad. (2022). 29, 896-907] is provided.

Studying membrane proteins with MicroED.

The structural investigation of biological macromolecules is indispensable in understanding the molecular mechanisms underlying diseases. Several structural biology techniques have been introduced to unravel the structural facets of biomolecules. Among these, the electron cryomicroscopy (cryo-EM) method microcrystal electron diffraction (MicroED) has produced atomic resolution structures of important biological and small molecules. Since its inception in 2013, MicroED established a demonstrated ability for solving structures of difficult samples using vanishingly small crystals. However, membrane proteins remain the next big frontier for MicroED. The intrinsic properties of membrane proteins necessitate improved sample handling and imaging techniques to be developed and optimized for MicroED. Here, we summarize the milestones of electron crystallography of two-dimensional crystals leading to MicroED of three-dimensional crystals. Then, we focus on four different membrane protein families and discuss representatives from each family solved by MicroED.

X-ray computerized microtomography and confocal Raman microscopy as complementary techniques to conventional imaging tools for the microstructural characterization of Cheddar cheese.

This study explored the use of X-ray computerized microtomography (micro-CT) and confocal Raman microscopy to provide complementary information to well-established techniques, such as confocal laser scanning microscopy (CLSM), for the microstructural characterization of cheese. To evaluate the potential of these techniques, 5 commercial Cheddar cheese samples, 3 with different ripening times and 2 with different fat contents, were analyzed. Confocal laser scanning microscopy was particularly useful to describe differences in fat and protein distribution, especially between the 2 samples with different fat contents. The quantitative data obtained through image analysis correlated well with the nutritional information provided in the product labels. Conversely, micro-CT was more advantageous for studying the size and spatial distribution of microcrystals present within the cheese matrix. Two types of microcrystals were identified that differed in size, shape, and X-ray attenuation. The smallest, with a diameter of approximately 10 to 20 µm, were more abundant in the samples and presented a more uniform roundish shape and higher X-ray attenuation. Larger and more heterogeneous crystals with diameters reaching 50 µm were also observed in scarcer numbers and showed lower X-ray attenuation. Confocal Raman microscopy was useful not only for identifying the distribution of all these components but also allowed comparing the presence of micronutrients such as carotenoids in the cheeses and provided compositional information on the crystals detected. Small and large crystals were identified as calcium phosphate and calcium lactate, respectively. Overall, using micro-CT, confocal Raman microscopy, and CLSM in combination generated novel and complementary information for the microstructural and nutritional characterization of Cheddar cheese. These techniques can be used to provide valuable knowledge when studying the effect of milk composition, processing, and maturation on the cheese quality attributes.

Tunable Triplet-Mediated Multicolor Lasing from Nondoped Organic TADF Microcrystals.

Thermally activated delayed fluorescent (TADF) emitters have received great attention in organic light-emitting diodes and laser diodes because of high exciton utilization efficiency and low optical loss caused by triplets. However, the direct observation of lasing emission from nondoped TADF microcrystals has yet to be reported. Here, we demonstrated a three-color (green, yellow, and red) microlaser from three nondoped TADF microcrystals with well-controlled geometries. The temperature-dependent dynamic analyses testify that the regenerated singlets which originated from the reverse intersystem crossing process at room temperature are beneficial for population inversion and reduce triplet-absorption/annihilation optical loses, together resulting in thermally activated lasing actions. Thanks to single-crystalline structures of TADF emitters, the relationship between triplet-harvesting capability and the molecular structure was systematically investigated. The results not only offer rational design of pure TADF gain materials but also provide guidance for the high-performance electrically driven organic solid-state lasers and multicolor laser integration.

A Hydrophobic Sisal Cellulose Microcrystal Film for Fire Alarm Sensors.

At present, environmentally friendly biobased flexible films are of particular interest as next-generation fireproof packaging and sensor materials. To reduce the moisture uptake and fire risks induced by hygroscopic and flammable biobased films, we report a simple and green approach to develop a hydrophobic, flame-retardant composite film with synergetic benefit from soy protein isolate (SPI), sisal cellulose microcrystals (MSF-g-COOH), graphene nanosheets (GN), and citric acid (CA). Compared with SPI/MSF-g-COOH composite films, the as-prepared SPI/MSF-g-COOH/CA/GN composite films have significantly improved water resistance and can maintain excellent physical structure and good electrical conductivity in an ethanol flame. This work opens a pathway for the development of novel fire-retardant fire alarm biosensors.

Unraveling the Electrical and Magnetic Properties of Layered Conductive Metal-Organic Framework With Atomic Precision.

This paper describes structural elucidation of a layered conductive metal-organic framework (MOF) material Cu3 (C6 O6 )2 by microcrystal electron diffraction with sub-angstrom precision. This insight enables the first identification of an unusual π-stacking interaction in a layered MOF material characterized by an extremely short (2.73 Å) close packing of the ligand arising from pancake bonding and ordered water clusters within pores. Band structure analysis suggests semiconductive properties of the MOF, which are likely related to the localized nature of pancake bonds and the formation of a singlet dimer of the ligand. The spin of CuII within the Kagomé arrangement dominates the paramagnetism of the MOF, leading to strong geometrical magnetic frustration.

Effect of sex and genotype of the host on the anthelmintic efficacy of albendazole microcrystals, in the CBi-IGE Trichinella infection murine model.

Albendazole (ABZ) is an anthelmintic pharmaceutical commonly used in the treatment of nematode infections. It is a Class II drug poorly water-soluble, with very low bioavailability, a feature particularly limiting to treat the trichinellosis chronic phase. Microcrystals obtained by controlled precipitation using hydroxyethyl cellulose and chitosan have previously been shown to improve ABZ biopharmaceutical properties. This investigation aimed to test the systems' in vivo efficacy in the CBi-IGE murine model of Trichinella spiralis infection in the infection's different phases and parasite' stages. Treatment in the enteral phase led to a 90% decrease in the larval muscle load, probably due to its effect on T. spiralis female fecundity. Both microcrystal systems given in the migratory phase halved muscle load in males, a response not observed in females. The chitosan-based microcrystals proved to be the best when administered in the chronic phase of the infection ­ an increased proportion of L1 dead larvae was found compared to controls, except in CBi+-treated females. Males and females from the highly susceptible CBi+ line presented a significantly different treatment response in this phase. In vivo efficacy depended on the host genotype and sex and was related to the parasite cycle stage in which the formulations were administered.

60-nt DNA Direct Detection without Pretreatment by Surface-Enhanced Raman Scattering with Polycationic Modified Ag Microcrystal Derived from AgCl Cube.

Direct detection of long-strand DNA by surface-enhanced Raman scattering (SERS) is a valuable method for diagnosis of hereditary diseases, but it is currently limited to less than 25-nt DNA strand in pure water, which makes this approach unsuitable for many real-life applications. Here, we report a 60-nt DNA label-free detection strategy without pretreatment by SERS with polyquaternium-modified Ag microcrystals derived from an AgCl cube. Through the reduction-induced decomposition, the size of the about 3 × 3 × 3 µm3 AgCl cube is reduced to Ag, and the surface is distributed with the uniform size of 63 nm silver nanoparticles, providing a large area of a robust and highly electromagnetic enhancement region. The modified polycationic molecule enhances the non-specific electrostatic interaction with the phosphate group, thereby anchoring DNA strands firmly to the SERS enhanced region intactly. As a result, the single-base recognition ability of this strategy reaches 60-nt and is successfully applied to detect thalassemia-related mutation genes.

Liquid chromatography tandem mass spectrometry method for determination of fulvestrant in rat plasma and its application to pharmacokinetic studies of a novel fulvestrant microcrystal.

Fulvestrant ('Faslodex'), an estrogen receptor antagonist, is available for the treatment of advanced breast cancer. The oil-based vehicle of Faslodex can lead to various adverse effects. A novel fulvestrant microcrystal (aqueous suspension) was developed in this study to eliminate these adverse effects. A sensitive and robust liquid chromatography tandem mass spectrometry method was developed and validated for the determination of fulvestrant in rat plasma using supported-liquid extraction. The separation of fulvestrant was achieved on an Agilent SB-C18 column (2.1 × 50 mm, 3.5 µm) with isocratic elution using fulvestrant-d3 as internal standard. Mass spectrometric detection was conducted in negative multiple reaction monitoring mode. Ion transitions were at m/z 605.5 → 427.5 for fulvestrant and m/z 608.5 → 430.5 for fulvestrant-d3. The excellent linearity was demonstrated over the range 0.05-100.0 ng/ml (r2 = 0.99). The lower limit of quantitation was 0.05 ng/ml, which was superior to that reported in literature The method validation was evaluated by selectivity, accuracy, precision, recovery and matrix effect in agreement with the US Food and Drug Administration guidance. The method was successfully applied to a pharmacokinetic study of a novel fulvestrant microcrystal in rats after intramuscular administration. It revealed that the rate of absorption increases and the extent of absorption is constant with a decrease in microcrystal diameter.