Journal of microscopy最新文献

The wavenumber nonlinearity leads to blurred reconstructed images in spectral-domain optical coherence tomography (SDOCT). In this work, a wavenumber-linearisation method without calibration devices is presented, based on the fact that the difference between the phases of adjacent peak and valley points is equal to $�\pi$. The theoretical model is derived, and the efficacy of the method was proven by acquiring SDOCT data from TiO₂ phantom and zebrafish. The results exhibit the superior performance of our method. Compared with the linear phase-based method, the resolution could be improved at least a factor of 2. Compared with the polynomial fitting method, the resolution could also be improved by nearly half.

In single-molecule microscopy, a big question is how precisely we can estimate the location of a single molecule. Our research shows that by using iterative localisation microscopy and factoring in the prior information, we can boost precision and reduce the number of photons needed. Leveraging the Van Trees inequality aids in determining the optimal precision achievable. Our approach holds promise for wider application in discerning the optimal precision across diverse imaging scenarios, encompassing various illumination strategies, point spread functions and overarching control methodologies.

Flow or collective movement is a frequently observed phenomenon for many cellular components including the cytoskeletal proteins actin and myosin. To study protein flow in living cells, we and others have previously used spatiotemporal image correlation spectroscopy (STICS) analysis on fluorescence microscopy image time series. Yet, in cells, multiple protein flows often occur simultaneously on different scales resulting in superimposed fluorescence intensity fluctuations that are challenging to separate using STICS. Here, we exploited the characteristic that distinct protein flows often occur at different spatial scales present in the image series to disentangle superimposed protein flow dynamics. We employed a newly developed and an established spatial filtering algorithm to alternatively accentuate or attenuate local image intensity heterogeneity across different spatial scales. Subsequently, we analysed the spatially filtered time series with STICS, allowing the quantification of two distinct superimposed flows within the image time series. As a proof of principle of our analysis approach, we used simulated fluorescence intensity fluctuations as well as time series of nonmuscle myosin II in endothelial cells and actin-based podosomes in dendritic cells and revealed simultaneously occurring contiguous and noncontiguous flow dynamics in each of these systems. Altogether, this work extends the application of STICS for the quantification of multiple protein flow dynamics in complex biological systems including the actomyosin cytoskeleton.

This study investigates the influence of the sample inherent temperature on the line-scan profile for a silicon trapezoid line with different sidewall angles by Monte–Carlo simulation. This study demonstrates that the profile varies with temperature, particularly focusing on the ‘shoulder’, which becomes more pronounced with larger sidewall angles. The contrast of the secondary electron profile increases at low primary electron energy but decreases at relatively high PE energy as the temperature rises. The trend of the backscattering electron profile is similar but less noticeable. The underlying mechanism is discussed in detail. This study has potential to provide valuable insights into thermometry in nanostructures using SEMs.

The structural nature and geometry, as well as the lattice-relative orientation, of an arrangement of crystal defects in a highly textured Eu²⁺-doped composite of two alkali-halide solid solutions was studied by epifluorescence microscopy (EFM) using the doping ion as a fluorochrome. A three-dimensional reconstruction and a skeleton type model, as built from a sequence of EFM images of different optical cross-sections of this arrangement, are presented. Structurally, this arrangement is a quadruple node (QN) of triple junctions of grain boundaries. The QN core geometry is that of a tetragonal tristetrahedron (TTTH), centred at the QN site, whose tetrahedron vertices and edges are on the QN triple junctions and grain boundaries, respectively, whereas the tristetrahedron tetragonal axis is nearly parallel to the lattice [001]-axis. The measured values of the angles between triple junctions and between the grain boundaries forming them are reported. The distinct chemical compositions of the composite solid solutions are discussed to be responsible, in last instance, for the tristetrahedron departure from a cubic configuration. Collaterally, certain families of translationally periodic almost-parallel (TPAP)-wall-like regions which consist of TPAP-columns of TPAP-spindle-like singularities, as well as certain zigzag arrays of columns of this like, existing into the QN grains, are reported to be observed. Three-dimensional reconstructions of typical individuals of these families and arrays as well as of their constituent parts are presented and geometrically analysed. These families and arrays are discussed to be families of tilt subboundaries, whose constituent dislocations are decorated by cylindrical second-phase europium di-halide precipitates, and regularly faceted tilt subboundaries, respectively. Crystal growing and sample preparation, composite structural characterisation by powder and single-slab X-ray diffraction (PXRD and SSXRD, respectively), microscopy and fluorescence-cube unit optics, image processing, electronic three-dimensional reconstruction and measuring methodologies, are all described in detail.

Nanoporous gold electrodes are of great interest in electroanalytical chemistry, because of their unusual activity and large surface area. The electrochemical activity can be further improved by coating with molecular catalysts such as the tetraruthenated cobalt-tetrapyridylporphyrazines investigated in this work. The plasmonic enhancement of the scattered light at the nanoholes and borders modifies the electrode's optical characteristics, improving the transmission through the surface-enhanced Raman scattering (SERS) effect. When monitored by hyperspectral dark-field and confocal Raman microscopy, this effect allows probing of the porphyrazine species at the plasmonic nanholes, improving the understanding of the chemically modified gold electrodes.

Immunohistochemistry (IHC) and immunofluorescence (IF) are crucial techniques for studying cardiac physiology and disease. The accuracy of these techniques is dependent on various aspects of sample preparation and processing. However, standardised protocols for sample preparation of tissues, particularly for fresh-frozen human left ventricle (LV) tissue, have yet to be established and could potentially lead to differences in staining and interpretation. Thus, this study aimed to optimise the reproducibility and quality of IF staining in fresh-frozen human LV tissue by systematically investigating crucial aspects of the sample preparation process. To achieve this, we subjected fresh-frozen human LV tissue to different fixation protocols, primary antibody incubation temperatures, antibody penetration reagents, and fluorescent probes. We found that neutral buffered formalin fixation reduced image artefacts and improved antibody specificity compared to both methanol and acetone fixation. Additionally, incubating primary antibodies at 37°C for 3 h improved fluorescence intensity compared to the commonly practised 4°C overnight incubation. Furthermore, we found that DeepLabel, an antibody penetration reagent, and smaller probes, such as fragmented antibodies and Affimers, improved the visualisation depth of cardiac structures. DeepLabel also improved antibody penetration in CUBIC cleared thick LV tissue fragments. Thus, our data underscores the importance of standardised protocols in IF staining and provides various means of improving staining quality. In addition to contributing to cardiac research by providing methodologies for IF, the findings and processes presented herein also establish a framework by which staining of other tissues may be optimised.