Journal of microscopy最新文献

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.

We present the analytical solution to the diffraction integral that describes the Rayleigh length for a focused Gaussian beam with any value of a spherical truncating aperture. This exact solution is in precise agreement with numerical calculations for the light distribution in the near focal area. The solution arises under assumption of the paraxial approximation, which also provides the basis for the classical Rayleigh length definition. It will be shown that the non-paraxial regime can be included by adding an empirical term (C_np) to the solution of the diffraction integral. This extends the validity of the expression to high numerical apertures (NA) up to n times 0.95, with n being the refractive index of the immersion medium. Thus, the entire practical range of NA, encountered in optical microscopy, is covered with a calculated error of less than 0.4% in the non-paraxial limit. This theoretical result is important in the design of optical instrumentation, where overall light efficiency in excitation and detection and spatial resolution must be optimised together.

Plasmodium sporozoites are the highly polarised and motile forms of the malaria parasite transmitted by mosquitoes to the vertebrate hosts. Sporozoites use myosin motors to generate retrograde flow of actin filaments. These are linked to plasma membrane spanning adhesins, which in turn bind to the extracellular environment, resulting in forward directed gliding motility. The gliding motility machine of sporozoites leads to high speeds in the range of micrometres per second, which are essential for efficient migration in the skin. Yet, it is not clear how the individual parts of the machinery work together to generate force during migration. Sporozoites are elongated and curved cells and move on circular tracks in vitro. Sporozoites lacking the adhesin thrombospondin-related anonymous protein (TRAP) like protein, TLP, can still migrate in the skin, but at a lower level. TLP lacking sporozoites generate a lower force on the dorsal (nonsubstrate facing) surface as measured by laser tweezers. Here we use traction force microscopy to investigate motile sporozoites and the forces they produce during migration on their ventral surface. Both wild type and tlp(-) sporozoites show distinct foci of force generation, but tlp(-) sporozoites generating overall lower forces. Our findings demonstrate that TLP is an important element of the force-generating machinery during sporozoite gliding motility.

Electron ptychography provides a promising avenue towards dose-efficient, high-resolution materials characterisation. Prior work demonstrates the feasibility of this approach, but an overarching view on the reliability of ptychographic images in low-dose scenarios is required. Here, we address this limitation with a systematic study of image clarity across dose, thickness and convergence semi-angle, on a range of materials science specimens. With the now widespread adoption of 4D-STEM and ptychographic imaging, the establishment of the practical parameter space in which one can anticipate a reliably interpretable phase image is urgently needed. In some cases, our parameter space exploration confirms high-resolution imaging at doses of 200 $�e^{-}$Å$�{}^{�-�2�}$.

In this study, we present a protocol to visualise, track and distinguish between two different binder components commonly used for batteries, styrene butadiene rubber (SBR) and sodium carboxymethyl cellulose (CMC), within a composite hard carbon electrode for sodium-ion batteries using a two-step staining method. The application of osmium tetroxide (OsO₄) vapour followed by uranyl acetate (UA) solution enables the staining of different functional groups and the individual tracing of SBR and CMC by energy dispersive X-ray spectroscopy (EDX) measurements using the osmium (Os) and uranium (U) content. This staining procedure and the filling of the pore space with conductive platinum carbon (PtC) composite via local electron-beam-induced deposition (EBID) results in an excellent contrast for all components of the electrode material. The tracking and visualisation of the binder components are demonstrated with secondary electron (SE) imaging and EDX mappings at focused ion beam (FIB) prepared facets as well as with focused ion beam/scanning electron microscopy (FIB/SEM) tomography.

LAY DESCRIPTION: In this study, a sample preparation protocol for hard carbon (HC) composite electrode material is presented which allows to clearly distinguish between the HC particle and the two binder components, styrene butadiene rubber (SBR) and sodium carboxymethyl cellulose (CMC) in focused ion beam/scanning electron microscopy (FIB/SEM) tomography and energy dispersive X-ray spectroscopy (EDX) measurements. For that, the material was stained with osmium tetroxide (OsO₄) and uranyl acetate (UA) and pore space was locally filled with electron-beam-induced deposition (EBID) of platinum carbon (PtC).

The axial positioning accuracy in confocal microscopy measurements is determined by the peak extraction algorithm of the intensity response curve. Existing peak extraction algorithms struggle to balance solution precision and computational efficiency, making it difficult to achieve online measurement. This paper proposes a fast Gaussian fitting method that transforms the nonlinear iterative fitting problem into a linear solution problem through Gaussian function linearisation, significantly improving computational efficiency. Experimental validation shows that the difference in solution accuracy between the fast Gaussian method and the traditional fitting method is no more than 3 nm, with a computational efficiency increasing at least 144 times.