Journal of microscopy最新文献

Integrated Centre-of-Mass (iCOM) is a widely used phase-contrast imaging method based on Centre-of-Mass (COM), which makes use of a 4D Scanning Transmission Electron Microscopy (STEM) dataset using an in-focus probe. In this paper, we introduce a novel approach that combines Single-Side Band (SSB) ptychography with COM and iCOM, termed Side Band masked Centre-of-Mass (SBm-COM) and integrated Centre-of-Mass (SBm-iCOM) which is applicable to weak-phase objects. This method compensates for residual aberrations in 4DSTEM datasets while also reducing the noise contribution up to the $��2�\alpha �$ resolution limit. The aberration compensation and noise filtering features make the SBm-(i)COM suitable for samples that are difficult to focus or those that require minimal electron fluence. SBm-iCOM transfers the same information as SSB ptychography but results in an intrinsic transfer function that enhances low-frequency information.

The endoplasmic reticulum (ER) is a highly dynamic organelle that undergoes significant morphological alterations in response to cellular stress. While conventional transmission electron microscopy (TEM) has provided valuable insights into these changes, such as the formation of crystalloid-ER and ER whorls, obtaining comprehensive three-dimensional (3D) information on these large structures within their cellular context has remained a challenge. To overcome these limitations, this study introduces an innovative application of dual-axis scanning transmission electron microscopy (STEM) tomography to investigate ER morphology under stress conditions in human embryonic kidney (HEK) cells overexpressing the cation channel polycystin-2 (PC-2). Benefitting from high-resolution, increased depth-of-focus, and reduced aberrations, STEM tomography enabled the detailed 3D reconstruction of large cellular subvolumes, providing unprecedented views of stress-induced ER structures. Our findings reveal distinct ultrastructural details of both crystalloid-ER and ER whorls. Crystalloid-ER exhibited a tubular architecture with potential interconnectedness, while ER whorls displayed a lamellar organisation and distinct membrane curvature. We observed the co-occurrence of these distinct smooth ER (sER) morphotypes within the same cell, yet they remained spatially separated, suggesting potential functional specialisation. Furthermore, we identified direct membrane contacts in mixed morphotypes, hinting at a shared origin or dynamic relationship between these structures. The study also elucidated the interactions of these organised smooth ER (OSER) structures with other organelles, such as mitochondria (MAM sites) and vesicles. In summary, the presented ultra-structural insights have a significant impact on our understanding of stress-related ER morphology changes. The ability to visualise the intricate 3D architecture and spatial relationships of these structures provides novel perspectives on the ER's adaptive responses to stress, including potential roles in lipid and protein biosynthesis and intracellular communication. These findings underscore the power of dual-axis STEM tomography for elucidating complex organellar organisation and dynamics in their native cellular context.

To address the critical need for investigating proton radiation effects on living cells in space environments and deciphering biological mechanisms underlying low-dose cumulative radiation effects, this study developed a microbeam irradiation microscopy platform. The system integrates a 10 MeV proton accelerator with a vertical microbeam line design. An ultrafast single-proton counting and radiation synchronisation control module-employing proton–photon–electron conversion and high-speed photoelectric circuitry achieve deterministic irradiation control with an end-to-end operational delay of 273.5 ns. Coupled with wide-field and confocal fluorescence microscopy, the platform enables real-time in situ observation during quantitative cellular irradiation, facilitating mechanistic studies of radiation-induced damage patterns and signal transduction in low-dose scenarios. Experimental validation using human embryonic kidney 293T cells demonstrated successful simulation of space radiation environments: dose-dependent DNA double-strand breaks (visualised via γ-H2AX foci) and radiation-induced bystander effects triggering damage propagation. These results establish the platform as an indispensable tool for space radiation health risk assessment while providing foundational insights into microscale energy deposition dynamics for proton therapy research.

Investigations of biological samples often require sample transparency, which is achieved by embedding the sample in a high-refractive index (RI) medium to obtain a homogenous RI distribution in the sample, referred to as optical clearing (OC). Here, we introduce a method for designing embedding media with an increased RI by increasing molecular orbitals, which is achieved by replacing elements in molecules commonly used for OC with elements possessing a more pronounced polarisability. Briefly, we took the established embedding medium Glycerol and exchanged the OH-groups by Thiol-groups, resulting in an embedding medium with very similar properties, but with a higher refractive index. We describe a procedure—abbreviated PRIAMOS—to render biological samples transparent using an RI-matching liquid, which we refer to as pH-value and Refractive Index Adjustment by Mixing highly polarisable molecular Orbital Substances. We focus on optical clearing in three-dimensional tissue samples whilst preserving fluorescence of fluorescent labels. The clearing procedure requires 2–3 days, yielding highly transparent samples, preserving the fluorescence of fluorescent proteins like the yellow fluorescent protein (YFP). This is demonstrated on mouse brain samples, imaged with standard confocal microscopy down to 1.6 mm depth, as well as on kidney samples. Mixtures of monothioglycerol, dithioglycerol and tributylamine achieve RI values between 1.52 and 1.57, and an acidity equivalent to pH values between 5 and 8. Our PRIAMOS approach can serve as a guideline for optimising optical clearing protocols.

This manuscript presents a comparative analysis of two software packages, MC X-ray and PENELOPE, focusing on their accuracy and efficiency in simulating k-ratios for binary compounds and comparing their spectra with experimental data for pure elements and compounds. Based on the Pouchou database, MC X-ray slightly outperforms PENELOPE in k-ratio calculations, achieving a root mean square error (RMSE) of 2.71% with a standard deviation of 0.027, compared to 2.87% with a standard deviation of 0.053. Discrepancies between the two programs emerge at lower beam energies (3 and 5 keV) when comparing simulated spectra with experimental data; however, at higher energies (20 and 30 keV), both software packages exhibit consistent and reliable performance across a range of atomic numbers. While both tools are effective for analysing homogeneous bulk samples, MC X-ray offers significant advantages in processing speed and user-friendliness. This study underscores the strengths and limitations of each package, providing valuable insights for researchers engaged in X-ray simulation and microanalysis.

We design and implement an original experimental platform resting on Atomic Force Microscopy (AFM) to capture nanoscale insights into key characteristics of solid/water interfaces subject to freeze-thaw conditions. The work is motivated by the observation that freezing and thawing underpin a variety of processes in the context of, e.g., climate and material sciences or cryobiology. Despite their key role, fundamental processes driving freezing and thawing are still elusive and their direct documentation is still challenging. This primarily stems from operational difficulties in replicating these processes under laboratory conditions, as well as constraints of current technology in matching temporal and spatial scales at which these phenomena take place. Here, we propose an experimental strategy to control freezing at solid/water interfaces while maintaining the bulk water as liquid. Our platform favors operational simplicity and can be integrated with any tip-scanning AFM. The strength of our set-up is assessed upon experiments performed on Highly Oriented Pyrolytic Graphite (HOPG) as a model substrate.

Kinetoplastid parasites include several species. Trypanosoma brucei causes African sleeping sickness in humans and a wasting disease nagana in livestock. Trypanosoma cruzi is the causative agent of Chagas disease and Leishmania species cause leishmaniasis, which can present with visceral, cutaneous, or mucocutaneous symptoms. All kinetoplastids harbour specialised peroxisomes called glycosomes, so named because most of the glycolytic pathway that is cytosolic in other eukaryotes is localised to these organelles. Glycosomes lack DNA and are essential for parasite viability. Despite their name, glycosomes also house enzymes involved in diverse pathways, including the pentose phosphate pathway, ether lipid biosynthesis, purine salvage, and sugar nucleotide biosynthesis. The degree to which these biochemical pathways localise together within the same organelle or to different glycosome populations is unclear. Biochemical fractionations and imaging data strongly suggest that glycosomes are heterogeneous in composition and that even within a single parasite, there are different glycosome populations. Until recently, we lacked the technology to systematically characterise glycosome populations within parasites. Glycosome morphology, composition, and localisation have historically been studied using widefield fluorescence and electron microscopy (EM). While EM can resolve individual organelles, it is extremely low throughput and requires specialised expertise and equipment. Widefield fluorescence imaging is higher throughput and more accessible. However, the small size of T. brucei cells, which are ∼20 µM in length and 3-5 µM in width, and glycosomes (100 nm in diameter) place these organelles below the resolution limits of standard microscopy and require super-resolution techniques to be resolved. These resolution issues are compounded by the cytoplasm's crowded nature, making it hard to discern individual organelles from each other. To overcome this, we leveraged recent advances in super-resolution microscopy, including a method called Ultrastructure Expansion Microscopy (U-ExM) combined with confocal imaging and LIGHTNING™ deconvolution to optimise the resolution of individual glycosomes. We found that antibodies against two different glycosome marker proteins (aldolase and GAPDH) exhibit discrete staining patterns. This high-resolution approach also revealed that glycosome morphology varies between monomorphic parasites that cannot complete the lifecycle and pleomorphic parasites that can, and is dynamically influenced by extracellular conditions, such as glucose availability, underscoring the adaptability of T. brucei's compartmentalisation to environmental changes.