Journal of microscopy最新文献

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.

Accurate detection of mitosis is crucial in automated cell analysis, yet many existing methods depend heavily on deep learning models or complex detection techniques, which can be computationally intensive and error-prone, particularly when segmentation is incomplete. This study presents a novel unsupervised method for mitosis detection, leveraging the geometric properties of the Cassini oval to reduce computational costs and enhance robustness. Our approach integrates a newly developed deep learning model, MaxSigNet, for initial cell segmentation. We subsequently employ the Cassini oval in its single-ring mode to detect mother cells in the initial frame and switch to double-ring mode in subsequent frames to identify daughter cells and confirm mitosis events. The success of this method hinges on the presence of equal non-zero foci values in the mother cell and distinct non-zero foci values in the daughter cells, which indicate accurate mitosis detection. The method was evaluated across six datasets from four different cell lines, achieving perfect F1, Recall and Precision scores on four datasets, with scores of 96% and 85% on the remaining two. Comparative analysis demonstrated that our method outperformed similar approaches in F1 and Recall metrics. Additionally, the method showed substantial robustness to incomplete segmentation, with only a 20% average drop in F1 scores when tested with older segmentation methods like K-means, Felzenszwalb and Watershed. The proposed method offers a significant advancement in mitosis detection by leveraging the Cassini oval's properties, providing a reliable and efficient solution for automated cell analysis systems. This approach promises to enhance the accuracy and efficiency of cellular behaviour studies, with potential applications in various biomedical research fields.

Multi-slice electron ptychography has attracted significant interest in recent years, thanks to notable experimental successes in ultra-high resolution, depth-resolved imaging of atomic structure. However, the theoretical dependence of depth of field on experimental parameters is not well understood. In this paper we use simulated data to compare the depth of field of through focal annular-dark field and multi-slice electron ptychography over a range of acceleration voltages and convergence angles. We show that at both low convergence angle and at low electron energy, multi-slice ptychography has significantly improved depth of field over through focal ADF imaging.

The scanning helium microscope (SHeM) is a new technology that uses a beam of neutral helium atoms to image surfaces non-destructively and with extreme surface sensitivity. Here, we present the application of the SHeM to image bacterial biofilms. We demonstrate that the SHeM uniquely and natively visualises the surface of the extracellular polymeric substance matrix in the absence of contrast agents and dyes and without inducing radiative damage.

Fluorescence lifetime imaging microscopy (FLIM) translates the duration of excited states of fluorophores into lifetime information as an additional source of contrast in images of biological samples. This offers the possibility to separate fluorophores particularly beneficial in case of similar excitation spectra. Here, we demonstrate the distinction of fluorescent molecules based on FLIM phasor analysis, called lifetime separation, in live-cell imaging using open-source software for analysis. We showcase two applications using Caenorhabditis elegans as a model system. First, we separated the highly spectrally overlapping fluorophores mCherry and mKate2 to distinctively track tagged proteins in six-dimensional datasets to investigate cell division in the developing early embryo. Second, we separated fluorescence of tagged proteins of interest from masking natural autofluorescence in adult hermaphrodites. For FLIM data handling and workflow implementation, we developed the open-source plugin napari-flim-phasor-plotter to implement conversion, visualisation, analysis and reuse of FLIM data of different formats. Our work thus advances technical applications and bioimage data management and analysis in FLIM microscopy for life science research.

Nanoscale optical imaging has unlocked unprecedented opportunities for exploring the structural, electronic, and optical properties of low-dimensional materials with spatial resolutions far beyond the diffraction limit. Techniques such as tip-enhanced, and tip-assisted photoluminescence (TEPL and TAPL), as well as scattering-type scanning near-field optical microscopy (s-SNOM) offer unique insights into local strain distributions, exciton dynamics, and dielectric heterogeneities that are inaccessible through conventional far-field approaches, however their combination within the same setup remains challenging. Here we present the realisation of correlative TEPL/TAPL and s-SNOM measurements within a single side-illuminated near-field optical microscope. We address the key experimental challenges inherent to the side-illumination geometry, including precise laser focus alignment, suppression of far-field background signals, and the mitigation of competing scattering pathways. Utilising monolayer WSe₂ as a model system, we demonstrate correlative imaging of material topography, strain-induced photoluminescence shifts, and dielectric function variations. We visualise nanoscale heterogeneities on a bubble-like structure, highlighting the complementary information from TAPL and s-SNOM. This correlative approach bridges the gap between nanoscale optical spectroscopy and near-field imaging, offering a powerful tool for probing local strain, doping, exciton behaviour, and dielectric inhomogeneities in low-dimensional materials.

Etch-pitting replication is a classical method to characterise the microstructure of ice crystals. In this method, a solution of polyvinyl formal (Formvar) is applied to a polished surface of ice. A plastic film, created after the solvent is dried, ‘replicates’ microstructural features of the ice. By examining the replica film, we can identify the orientation of crystals and existence of dislocations in ice. However, with the recent rise of advanced techniques such as cryo-EBSD (electron backscatter diffraction) analyses, this classical method has been left in the shadows, especially from the perspective of quantification of microstructural features in polycrystalline ice. In this study we revive and thoroughly re-examine the utility of the replication method to quantify crystal orientations and dislocation density of ice. We applied our optimised protocols of the replication method to several laboratory-fabricated and natural-glacier polycrystalline ice samples with various types of crystal preferred orientation (CPO) and various levels of strain. Using high-resolution scanning electron microscope (SEM) images of the obtained replica films, we quantified the extent of CPO and dislocation density of these ice samples. Our results of CPO patterns and dislocation density show good agreement with cryo-EBSD results from the same ice samples or samples at a similar strain level. Although further improvements are needed to make the present method more efficient, our results show promise for using this method to easily, quickly, and affordably quantify microstructural features in polycrystalline ice and to help interpret deformation mechanism of ice.