Journal of microscopy最新文献

Mitochondria are double-membrane organelles whose architecture enables ATP (Adenosine Triphosphate) production, redox signalling, calcium homeostasis, and apoptosis. Visualisation of mitochondria requires imaging technologies across spatial and temporal scales. Conventional fluorescence microscopy techniques, such as wide-field, confocal, spinning-disk, and light-sheet microscopy, enable the real-time observation of mitochondrial networks and dynamics in live cells. Super-resolution methods, including structured illumination microscopy (SIM), stimulated emission depletion microscopy (STED), photoactivated localisation microscopy (PALM), stochastic optical reconstruction microscopy (STORM), and expansion microscopy, provide access to fine sub-mitochondrial structures, such as cristae, overcoming the diffraction limit. Additionally, proximity-based approaches such as FRET (Förster Resonance Energy Transfer), split-fluorescent proteins, and proximity ligation assays allow researchers to probe sub-compartmental interactions and organelle contact sites with nanometre-level sensitivity. Electron microscopy (EM) complements optical techniques by offering near-molecular resolution of mitochondrial ultrastructure, including membranes, cristae, and inter-organelle interfaces. In this review, we comprehensively examined the principles, capabilities, and limitations of these diverse imaging modalities, with a focus on recent advances. We highlight the development of novel fluorescent probes, integrated correlative techniques, and computational analysis pipelines to expand the utility of mitochondrial imaging. By placing these innovations in historical and theoretical contexts, we aim to clarify how each method works and why it is suited to biological questions. Finally, we explore how mitochondrial imaging has revolutionised our understanding of physiology and pathology.

The microstructure distribution of multiphase porous media has important effects on its macroscopic properties. In this paper, we propose a framework for multiphase reconstruction based on entropy statistical descriptor, which is used as morphological information to perform on two-dimensional (2D) and three-dimensional (3D) reconstruction. The accuracy of the 2D reconstruction is evaluated using the lineal-path function and two-point cluster function, which reflect the connectivity of the microstructure. From the result of the 2D reconstructions, it is noted that the reconstructed three-phase microstructures have the similar morphological information with that of the original images and their lineal-path function and two-point cluster function are closely similar to each other. For the reconstructed 3D microstructures, the accuracy of the reconstructions is also quantified by the lineal-path function and two-point cluster function, which reflect the comprehensive information of the 3D connectivity of the system. The comparison of the lineal-path function and two-point cluster function between all the slices of the reconstructed 3D structures in three directions and that of the reference images shows that the proposed method can capture the prominent features of the original images. The lineal-path function and two-point cluster function of the reconstructed 3D structures are also compared with that of their original 3D structures, which shows that the reconstructed 3D structures have the similar distribution with that of the original 3D structures. This indicates that our proposed method has the ability to capture salient morphological information of multiphase system.

Cellulose materials are suitable to replace plastic in food packaging. They are hydrophilic and may have poor barrier properties that affect the shelf life of the food due to the migration of contaminants. The wet lamination of microfibrillated cellulose films on cellulose materials appears as a promising way to improve their barrier properties by forming a bilayer material. These barrier properties depend on the microstructural properties (porosity, pores connectivity, specific surface area or contact surface area between the two layers) of both layers, notably the film, which are poorly known. Therefore, a multiscale approach is proposed to estimate such microstructural parameters by combining three 3D imaging methods: synchrotron X-ray micro-/nanotomography and FIB-SEM tomography. The 3D microstructure of two different bilayer materials obtained with two Microfibrillated Cellulose (MFC) grades is investigated. For the first time, a full 3D representation of such material is presented. Regardless of the scale under consideration, our results showed that both films present a dense structure with very low porosity and no pore connectivity along the thickness. The MFC film produced with the smallest MFC fibrils led to a more homogeneous and less porous layer with a larger contact surface to the paper.

We compare three different methods of X-ray analysis in a scanning electron microscope (SEM): energy-dispersive X-ray spectroscopy (EDX), wavelength-dispersive X-ray spectroscopy (WDX) and micro X-ray fluorescence (μXRF). These methods are all applied to the same gallium arsenide (GaAs) wafer with a 0.8 nm layer of indium arsenide (InAs) on top. All methods allow detection and quantification of the indium L-line intensity from the thin InAs layer. EDX is the easiest to perform, WDX is the most sensitive and μXRF a novel technique where a poly-capillary optics is used to focus an X-ray beam from a high-voltage X-ray tube onto a small spot several micrometres wide and the characteristic X-rays produced are detected by a solid-state silicon detector similar to that used in EDX. It is to our knowledge the first time a sub-nanometre layer is reliably detected and analysed using μXRF in an SEM.

Expansion microscopy (ExM) is a powerful high-resolution imaging technique that enhances the spatial resolution of conventional light microscopy by physically enlarging biological specimens by embedding and cross-linking them in a swellable polymer network. This review explores the combination of ExM with commonly used advanced fluorescence imaging modalities, including light sheet fluorescence microscopy (LSFM), stimulated emission depletion (STED), structured illumination microscopy (SIM), single-molecule localisation microscopy (SMLM), and computational super-resolution radial fluctuations (SRRF) to push the boundaries of achievable resolution in biological imaging. By integrating ExM with these optical and analytical approaches, researchers can visualise subcellular structures and molecular complexes with unprecedented clarity, enabling the study of intricate biological processes that are otherwise inaccessible with conventional light microscopy methods. The review covers the theoretical resolutions attainable with each combined technique, example biological questions they can address, and key considerations for optimising their use. Together, these advancements offer novel insights into nanoscale cellular and subcellular structures, opening new avenues for exploration in fields such as neuroscience, cancer research, and developmental biology.

Some biological systems exhibit nanoscale constructions to produce optical effects. This study utilises Atomic Force Microscopy (AFM) and Tip-Enhanced Raman Spectroscopy (TERS) to study the complex bionanometric structure of cicada wings. Topographical irregularities of the wings due to $�\alpha$-chitin nanopillars hinder the probe's approach to the sample, a crucial step in overcoming the light diffraction limit in TERS measurements. To mitigate this issue, graphene was deposited, promoting surface smoothing and ensuring a reliable TERS measurement. Combined analyses of AFM and TERS mapping revealed a significant enhancement of the graphene 2D band, particularly in the regions surrounding the nanometric pillars, while the characteristic $�\alpha$-chitin Raman peaks are evident on top of the pillars, clarifying details of how light passed through the material.

Localisation microscopy often relies on detailed models of point-spread functions. For applications such as deconvolution or PSF engineering, accurate models for light propagation in imaging systems with a high numerical aperture are required. Different models have been proposed based on 2D Fourier transforms or 1D Bessel integrals. The most precise ones combine a vectorial description of the electric field and accurate aberration models. However, it may be unclear which model to choose as there is no comprehensive comparison between the Fourier and Bessel approaches yet. Moreover, many existing libraries are written in Java (e.g., our previous PSF generator software) or MATLAB, which hinders their integration into deep learning algorithms. In this work, we start from the original Richards-Wolf integral and revisit both approaches in a systematic way. We present a unifying framework in which we prove the equivalence between the Fourier and Bessel strategies and detail a variety of correction factors applicable to both of them. Then, we provide a high-performance implementation of our theoretical framework in the form of an open-source library that is built on top of PyTorch, a popular library for deep learning. It enables us to benchmark the accuracy and computational speed of different models and allows for an in-depth comparison of the existing models for the first time. We show that the Bessel strategy is optimal for axisymmetric beams, while the Fourier approach can be applied to more general scenarios. Our work enables the efficient computation of a point-spread function on CPU or GPU, which can then be included in simulation and optimisation pipelines.

Expansion microscopy (ExM) enables superresolution imaging by embedding biological specimens in a swellable hydrogel, followed by optical clearing and physical expansion. Here we present a detailed protocol for applying ExM to embryonic stages of Paracentrotus lividus, a widely used model in developmental biology. Embryos at cleavage and gastrula stages, as well as pluteus larvae, were successfully expanded fourfold after proteinase digestion. Preexpansion Airyscan imaging provided only limited subcellular information due to autofluorescence, whereas post-expansion samples displayed markedly reduced background and resolved fine structures. To address distortions caused by the calcified skeleton in pluteus larvae, we incorporated a decalcification step with EDTA, which preserved morphology and enabled isotropic expansion. Distinct NHS ester dyes further allowed differential labelling of the fertilisation envelope and blastomeres, illustrating the versatility of this approach. Together, these adaptations establish a reproducible workflow for ExM in marine invertebrates, offering a valuable methodological resource and a foundation for future applications in developmental and environmental research.