Journal of microscopy最新文献

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.

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.

Neospora caninum is known to manipulate host cell organelles and recruit lipids for its survival. However, the impact of this lipid redistribution on host cell membranes remains poorly understood. This study used LAURDAN fluorescence, hyperspectral imaging, and phasor plot analysis to investigate how N. caninum modifies membrane order in Vero cells. The results revealed a significant decrease in host cell plasma and internal membrane order upon infection, suggesting that cholesterol is redistributed from the host plasma membrane to the parasitophorous vacuoles. To mimic cholesterol depletion, uninfected cells were treated with methyl-β-cyclodextrin (MBCD), which increased membrane fluidity. Conversely, replenishing infected cells with cholesterol-loaded MBCD restored membrane fluidity to levels lower than control cells, indicating cholesterol enrichment. These findings provide novel insights into how N. caninum modulates host cell membrane dynamics through lipid manipulation, potentially aiding its intracellular survival.

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.

Cryo-electron tomography (cryo-ET) has become a powerful tool for visualising cellular structures at sub-nanometer resolution in their near-native state, offering unique insights into the molecular architecture of diverse biological systems, including infectious agents and their interactions with host cells. This paper reviews key methodologies and recent advancements in cryo-ET, with a particular focus on sample preparation of protozoan parasites and host cells. Topics covered include photopatterning for cell positioning on EM grids, vitrification techniques, whole-cell imaging, and cryo-FIB milling followed by cryo-ET. The manuscript also addresses how these approaches are providing valuable structural information on pathogens and pathogen-host interactions, which are critical for understanding mechanisms of pathogenesis and the development of therapeutic strategies. Additionally, we examine the principles and practical considerations of the multistep workflow, highlighting innovations such as integrated fluorescence microscopy (iFLM) within cryo-FIB SEM systems for improved target identification and lamella positioning. Challenges such as ion beam damage, sample thickness constraints, and the need for greater workflow automation are also discussed as areas for future improvement. As cryo-ET continues to evolve and deliver transformative insights into the molecular architecture of life, it inspires great hope for the development of future therapies against infectious diseases. LAY DESCRIPTION: Cryo-electron tomography (cryo-ET) is a special type of microscopy that allows researchers to look at the inside of cells in 3D, almost as if a hologram of the cell in its natural state was generated. This technique reveals molecular structures inside cells, allowing scientists to better understand how molecules and cellular components work together. To obtain such detailed images, biological samples need to be thin and frozen very quickly so that they remain undamaged and close to their natural state. One recent breakthrough involves using a tool called cryo-focused ion beam scanning electron microscopy (cryo-FIB SEM), which allows a thin slice of a frozen sample to be collected and then analysed using cryo-ET. In addition, photopatterning of support surfaces are being used to place cells in a strategic position for cryo-FIB SEM, and improved plunge freezing and high-pressure freezing methods have been developed to better preserve samples. Together, these techniques make it easier to reproducibly prepare high-quality samples for cryo-ET. These innovations allow capturing clearer and detailed images of cells, tissues, and even entire small organisms. Cryo-ET has led to important discoveries in biology, such as how proteins and other molecules interact within cells at the sub-nanometre scale. This technique holds great promise for revealing how life works at a molecular level, understanding diseases, and discovering new drugs.

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.

Malaria is one of the deadliest infectious diseases in the world, annually responsible for over 400,000 deaths. It is caused by parasites of the genus Plasmodium, which undergo remarkable structural changes during their development within different cells across various hosts. An important approach to understand the structural basis of biochemical and physiological processes during Plasmodium infection has been the quantitative measurement of dimensional parameters obtained by different microscopy techniques. In this regard, sample preparation, particularly electron microscopy protocols that rely on room-temperature chemical fixation, has posed significant challenges, as it is known to produce artefacts such as shrinking, swelling and displacement of structures and osmolytes. In contrast, specimen immobilisation by cryofixation followed by freeze substitution minimises these artefacts and provides better sample preservation. Nevertheless, the composition of the freeze substitution medium may vary depending on the cell type, making it a critical factor for achieving optimal sample preparation. In this work, we optimised a freeze substitution protocol for the structural analysis of intraerythrocytic stages of the murine malaria models Plasmodium chabaudi and P. berghei. We tested different freeze substitution recipes, considering the biochemical composition of malaria membranes, and compared the results with those obtained through conventional chemical fixation. Overall, the results showed a significant improvement on the preservation of cell morphology and haemozoin crystals. Establishing an efficient and reproducible freeze substitution protocol for murine malaria models provides an important tool for advancing our understanding of the structural organisation of Plasmodium spp.

Apicomplexans, a large phylum of protozoan intracellular parasites, well known for their ability to invade and proliferate within host cells, cause diseases with major health and economic impacts worldwide. These parasites are responsible for conditions such as malaria, cryptosporidiosis, and toxoplasmosis, which affect humans and other animals. Apicomplexans exhibit complex life cycles, marked by diverse modes of cell division, which are closely associated with their pathogenesis. All the unique structural and evolutionary characteristics of apicomplexan parasites, the biology underlying life stage transitions, and the singular mechanisms of cell division alongside their associated biomedical relevance have captured the attention of parasitologists of all times. Traditional light and electron microscopy have set the fundamental foundations of our understanding of these parasites, including the distinction among their modes of cell division. This has been more recently complemented by microscopy advances through the implementation of superresolution fluorescence microscopy, and variants of electron microscopy, such as cryo-EM and tomography, revealing intricate details of organelles and cell division. Ultrastructure Expansion Microscopy has emerged as a transformative, accessible approach that enhances resolution by physically expanding samples isometrically, allowing nanoscale visualisation on standard light microscopes. In this work, we review the most recent contributions of U-ExM and its recent improvements and innovations, in providing unprecedented insights into apicomplexan ultrastructure and its associated mechanisms, focusing particularly on cell division. We highlight the power of U-ExM in combination with protein-specific labelling, in aiding the visualisation of long oversighted organelles and detailed insights into the assembly of parasite-specific structures, such as the conoid in Plasmodia, and the apical-basal axis in Toxoplasma, respectively, during new parasite assembly. Altogether, the contributions of U-ExM reveal conserved and unique structural features across species while nearing super resolution. The development of these methodologies and their combination with different technologies are crucial for advancing our mechanistic understanding of apicomplexan biology, offering new perspectives that may facilitate novel therapeutic strategies against apicomplexan-caused diseases.