Journal of microscopy最新文献

Electron backscatter diffraction (EBSD) has developed over the last few decades into a valuable crystallographic characterisation method for a wide range of sample types. Despite these advances, issues such as the complexity of sample preparation, relatively slow acquisition, and damage in beam-sensitive samples, still limit the quantity and quality of interpretable data that can be obtained. To mitigate these issues, here we propose a method based on the subsampling of probe positions and subsequent reconstruction of an incomplete data set. The missing probe locations (or pixels in the image) are recovered via an inpainting process using a dictionary-learning based method called beta-process factor analysis (BPFA). To investigate the robustness of both our inpainting method and Hough-based indexing, we simulate subsampled and noisy EBSD data sets from a real fully sampled Ni-superalloy data set for different subsampling ratios of probe positions using both Gaussian and Poisson noise models. We find that zero solution pixel detection (inpainting un-indexed pixels) enables higher-quality reconstructions to be obtained. Numerical tests confirm high-quality reconstruction of band contrast and inverse pole figure maps from only 10% of the probe positions, with the potential to reduce this to 5% if only inverse pole figure maps are needed. These results show the potential application of this method in EBSD, allowing for faster analysis and extending the use of this technique to beam sensitive materials.

Magnaporthe oryzae is the causal agent of rice blast, one of the most serious diseases affecting rice cultivation around the world. During plant infection, M. oryzae forms a specialised infection structure called an appressorium. The appressorium forms in response to the hydrophobic leaf surface and relies on multiple signalling pathways, including a MAP kinase phosphorelay and cAMP-dependent signalling, integrated with cell cycle control and autophagic cell death of the conidium. Together, these pathways regulate appressorium morphogenesis.The appressorium generates enormous turgor, applied as mechanical force to breach the rice cuticle. Re-polarisation of the appressorium requires a turgor-dependent sensor kinase which senses when a critical threshold of turgor has been reached to initiate septin-dependent re-polarisation of the appressorium and plant infection. Invasive growth then requires differential expression and secretion of a large repertoire of effector proteins secreted by distinct secretory pathways depending on their destination, which is also governed by codon usage and tRNA thiolation. Cytoplasmic effectors require an unconventional Golgi-independent secretory pathway and evidence suggests that clathrin-mediated endocytosis is necessary for their delivery into plant cells. The blast fungus then develops a transpressorium, a specific invasion structure used to move from cell-to-cell using pit field sites containing plasmodesmata, to facilitate its spread in plant tissue. This is controlled by the same MAP kinase signalling pathway as appressorium development and requires septin-dependent hyphal constriction. Recent progress in understanding the mechanisms of rice infection by this devastating pathogen using live cell imaging procedures are presented.

The idea that disease is caused at the cellular level is so fundamental to us that we might forget the critical role microscopy played in generating and developing this insight. Visually identifying diseased or infected cells lays the foundation for any effort to curb human pathology. Since the discovery of the Plasmodium-infected red blood cells, which cause malaria, microscopy has undergone an impressive development now literally resolving individual molecules. This review explores the expansive field of light microscopy, focusing on its application to malaria research. Imaging technologies have transformed our understanding of biological systems, yet navigating the complex and ever-growing landscape of techniques can be daunting. This review offers a guide for researchers, especially those working on malaria, by providing historical context as well as practical advice on selecting the right imaging approach. The review advocates an integrated methodology that prioritises the research question while considering key factors like sample preparation, fluorophore choice, imaging modality, and data analysis. In addition to presenting seminal studies and innovative applications of microscopy, the review highlights a broad range of topics, from traditional techniques like white light microscopy to advanced methods such as superresolution microscopy and time-lapse imaging. It addresses the emerging challenges of microscopy, including phototoxicity and trade-offs in resolution and speed, and offers insights into future technologies that might impact malaria research. This review offers a mix of historical perspective, technological progress, and practical guidance that appeal to novice and advanced microscopists alike. It aims to inspire malaria researchers to explore imaging techniques that could enrich their studies, thus advancing the field through enhanced visual exploration of the parasite across scales and time.

This article presents a qualitative, quantitative, and experimental analysis of optical coherence tomography (OCT) volumes obtained using different families of non-raster trajectories. We propose a multicriteria analysis to be used in the assessment of scan trajectories used in obtaining OCT volumetric point cloud data. The novel criteria includes exploitation/exploration ratio of the OCT data obtained, smoothness of the scan trajectory and fast preview of the acquired OCT data in addition to conventional criteria; time and quality (expressed as volume similarity rather than slice-by-slice image quality). The set of criteria proposed will be useful in assessing OCT scan trajectories for optimisation in various applications including robot assisted in vivo optical biopsy. We show in this paper that the rate of data acquisition is improved without degrading the OCT volume quality by scanning using non-raster trajectories (they are fast, smooth, and make the galvanometer scanners have less wear and tear). In particular, the rosette scan trajectory, which was the preferred non-raster trajectory, provided a balanced performance in having better clarity at the centre and periphery of the scanned object.

This short review discusses the recent developments in low-cost, high-resolution optoacoustic microscopy systems, integrating laser diodes for signal excitation, which are 20-40 times cheaper than the typically employed Q-switched nanosecond laser sources. The development of laser diode-based microscopes can substantially improve not only cost efficiency, but also multispectral capabilities, robustness, portability and overall imaging performance of the optoacoustic technique. To this end, we demonstrate relevant implementations in both time and frequency domain, highlighting their representative applications in biomedical research such as microvasculature imaging, oxygen saturation assessments, hybrid and multiview microscopy of model organisms and tissues and Doppler flow speed measurements. Finally, we analyse the benefits and limitations of each approach, identifying the respective application contexts where they achieve optimum performance.

Conventional optical microscopy imaging of obligate intracellular bacteria is hampered by the small size of bacterial cells, tight clustering exhibited by some bacterial species and challenges relating to labelling such as background from host cells, a lack of validated reagents, and a lack of tools for genetic manipulation. In this study, we imaged intracellular bacteria from the species Orientia tsutsugamushi (Ot) using five different fluorescence microscopy techniques: standard confocal, Airyscan confocal, instant Structured Illumination Microscopy (iSIM), three-dimensional Structured Illumination Microscopy (3D-SIM) and Stimulated Emission Depletion Microscopy (STED). We compared the ability of each to resolve bacterial cells in intracellular clumps in the lateral (xy) axis, using full width half-maximum (FWHM) measurements of a labelled outer membrane protein (ScaA) and the ability to detect small, outer membrane vesicles external to the cells. Comparing the techniques readily available to us (above), 3D-SIM microscopy, in combination with the shortest-wavelength dyes, was found overall to give the best lateral resolution. We next compared the ability of each technique to sufficiently resolve bacteria in the axial (z) direction and found 3D-STED to be the most successful method for this. We then combined this 3D-STED approach with a custom 3D cell segmentation and analysis pipeline using the open-source, deep learning software, Cellpose to segment the cells and subsequently the commercial software Imaris to analyse their 3D shape and size. Using this combination, we demonstrated differences in bacterial shape, but not their size, when grown in different mammalian cell lines. Overall, we compare the advantages and disadvantages of different super-resolution microscopy techniques for imaging this cytoplasmic obligate intracellular bacterium based on the specific research question being addressed.

The endoplasmic reticulum (ER) forms contact sites with the chloroplast. Exposing contact sites that contain both the chloroplast and the ER to localised high-fluence, wavelength specific, 405 nm violet light, hereinafter referred to as photostimulation, induces multiple, potentially interacting intra- and intercellular responses. The responses vary depending on the tissue type of the cell and the chloroplast. Photostimulating the ER-chloroplast contact sites in growing epidermal cells of the hypocotyl of Arabidopsis thaliana, produces a wave of cytoplasmic ionic calcium that traverses the cell, spreading radially to other cells around the circumference of the hypocotyl. A transient ER stress accompanies the calcium wave. These responses occur in older epidermal cells (5-8 days post-germination) with nonmotile chloroplasts tethered to the ER and the cell cortex but do not occur with motile or dividing chloroplasts. Dividing chloroplasts show a markedly different association with the ER, which forms a ring around the fission plane, similar to that of dividing mitochondria. Inhibition of calcium channels with lanthanum has no effect. Photostimulation of only the ER results in no ER stress and a calcium wave with a different spatiotemporal signature: delayed release and lower magnitude, with no accompanying ER stress response. Likewise, photostimulation of the chloroplast only, without the ER, produces no calcium wave or ER stress. General chloroplast photobleaching or restructuring caused by photostimulation is not the cause of this response; photostimulation with 488 nm of the same intensity and power as 405 nm photostimulation produces no change in cytosolic calcium levels. The pH of the ER decreases, indicating the involvement of ER ion transporters in the response. A wave of increased reactive oxygen species (ROS) in mitochondria and nuclei accompanies photostimulation. Together, these data support a model by which tethered ER-chloroplast contact sites constitute a unique subcellular photosensitive region and are part of an ER-mediated signalling network. Lay Abstract: The endoplasmic reticulum (ER) forms contact sites with the chloroplast. Shining violet (405 nm) light on the chloroplast with its associated ER produces a calcium wave through the cell that is communicated to other cells. This is correlated with a wave of transient denaturation of the luminal proteins of the ER (ER stress) and increased reactive oxygen species (ROS) in mitochondria. The wavelength dependence and precise cellular location of the light stimulation implies a novel way for plants to sense light. The movement of the response through the cell is consistent with the mediation of the response by a subcellular network, such as that formed by the ER.

Interference Reflection Microscopy (IRM) is an optical technique that relies on the interference between the reflected light from an incident beam as it passes through materials of different refractive indices. This technique has been successfully used to image microtubules, biologically important biofilaments with a diameter of 25 nm. However, it is often desirable to image both the microtubule and microtubule interacting proteins simultaneously. Here we present a simple modification to a standard multicolour total internal reflection fluorescence (TIRF) microscope that enables simultaneous high-speed IRM and single molecule TIRF imaging. Our design utilises a camera for each channel (IRM and TIRF) allowing independent optimisation of camera parameters for the two different modalities. We illustrate its application by imaging unlabelled microtubules and GFP-labelled end-binding protein EB1, which forms comets on the tips of polymerising microtubules. Our design is easily implemented, and with minimal cost, making it accessible to any laboratory with an existing fluorescence microscope.

Osteoblasts are the functional cells capable of bone formation in the bone microenvironment and play an important role in bone growth, development, and the maintenance of bone mass. The cells cultured in vitro are derived from preosteoblasts in tissues and possess the ability to divide and proliferate. Osteoblasts form the bone matrix by secreting collagen and other matrix proteins, which provides a foundation for the deposition of minerals such as calcium and phosphorus, ultimately resulting in the formation of hard bone tissue. Bone diseases affect the quality of life and the aging of the population. Bone diseases such as osteoporosis, fractures, bone tumours, and arthritis have a significant impact on quality of life, especially among the elderly population. These realities remind us that we should pay more attention to bone and joint health. Therefore, it is particularly important to study the imaging and characterisation of mechanical properties of bone cells, which provides a basis for the research of bone diseases in human beings.