细胞生物学

An improved correlative light and electron microscopy (CLEM) method has recently been introduced and successfully employed to identify and analyze protein inclusions in cultured cells as well as pathological proteinaceous deposits in postmortem human brain tissues from individuals with diverse neurodegenerative diseases. This method significantly enhances antigen preservation and target registration by replacing conventional dehydration and embedding reagents. It achieves an optimal balance of sensitivity, accuracy, efficiency, and cost-effectiveness compared to other current CLEM approaches. However, due to space constraints, only a brief overview of this method was provided in the initial publication. To ensure reproducibility and facilitate widespread adoption, the author now presents a detailed, step-by-step protocol of this optimized CLEM technique. By enhancing usability and accessibility, this protocol aims to promote broader application of CLEM in neurodegenerative disease research.

Matrix vesicles (MVs) represent a heterogeneous group of spherical membrane-bound extracellular vesicles in the range of 100–200 nm in diameter secreted by mineralizing osteoblasts. The initial synthesis of the amorphous calcium phosphate occurs within the confines of the intracellular MVs, which are capable of transporting P_i and Ca²⁺ into the MV lumen. Thus, understanding the initial process of MV-mediated mineralization is critical in developing better therapeutic strategies for various bone-related disorders such as osteoporosis and addressing ectopic calcification of soft tissues. Although various techniques and commercially available kits are now available for isolating MVs, isolating a pure population of MVs is challenging mainly because of their variable size and lack of consensus protein markers. This ultracentrifugation-based protocol ensures high purity of isolated MVs by removing other contaminated extracellular vesicles and cellular debris through sequential centrifugation steps but also allows downstream functional mineralization assays of the isolated MVs.

Cryo-electron microscopy (cryo-EM) is a powerful technique capable of investigating samples in a hydrated state, compared to conventional high-vacuum electron microscopy that requires samples to be completely dry. During the drying process, numerous features and details may be lost due to damage caused by dehydration. Cryo-EM circumvents these problems by cryo-fixing the samples, thereby retaining the intact and original features of hydrated samples. This protocol describes a step-by-step cryo-scanning electron microscopy (cryo-SEM) experimental procedure with Chlorella sorokiniana as the subject. By employing filter paper as the sample substrate, we propose a simple and reliable method for cryo-fixation and freeze-fracture of Chlorella sorokiniana in water suspension. The advantage of using filter paper as a substrate lies in its ability to support a thin film of sample, enabling a cold knife to make a cut effortlessly and produce a clean freeze-fractured surface for SEM investigation. By following the approach described in this protocol, both the internal structure and surface morphology of Chlorella sorokiniana can be easily resolved with high quality. This protocol is highly versatile and can be applied to samples dispersed in water or solvents, including cyanobacterial cells, algal cells, and any kind of sample that can be adsorbed onto filter paper.

In plants, the first interaction between the pollen grain and the epidermal cells of the stigma is crucial for successful reproduction. When the pollen is accepted, it germinates, producing a tube that transports the two sperm cells to the ovules for fertilization. Confocal microscopy has been used to characterize the behavior of stigmatic cells post-pollination [1], but it is time-consuming since it requires the development of a range of fluorescent marker lines. Here, we propose a quick, high-resolution imaging protocol using tabletop scanning electron microscopy. This technique does not require prior sample fixation or fluorescent marker lines. It effectively captures pollen grain behavior from early hydration (a few minutes after pollination) to pollen tube growth within the stigma (1 h after pollination) and is particularly efficient for tracking pollen tube paths.

For the analysis of cellular architecture during mitosis, nanometer resolution is needed to visualize the organization of microtubules in spindles. Here, we present a detailed protocol that can be used to produce 3D reconstructions of whole mitotic spindles in cells grown in culture. For this, we attach mammalian cells enriched in mitotic stages to sapphire discs. Our protocol further involves cryo-immobilization by high-pressure freezing, freeze-substitution, and resin embedding. We then use fluorescence light microscopy to stage select mitotic cells in the resin-embedded samples. This is followed by large-scale electron tomography to reconstruct the selected and staged mitotic spindles in 3D. The generated and stitched electron tomograms are then used to semi-automatically segment the microtubules for subsequent quantitative analysis of spindle organization. Thus, by providing a detailed correlative light and electron microscopy (CLEM) approach, we give cell biologists a toolset to streamline the 3D visualization and analysis of spindle microtubules (http://kiewisz.shinyapps.io/asga). In addition, we refer to a recently launched platform that allows for an interactive display of the 3D-reconstructed mitotic spindles (https://cfci.shinyapps.io/ASGA_3DViewer/).

Key features

• High-throughput screening of mitotic cells by correlative light and electron microscopy (CLEM).

• Serial-section electron tomography of selected cells.

• Visualization of mitotic spindles in 3D and quantitative analysis of microtubule organization.

Graphical overview

Combining two different plants together through grafting is one of the oldest horticultural techniques. In order to survive, both partners must communicate via the formation of de novo connections between the scion and the rootstock. Despite the importance of grafting, the ultrastructural processes occurring at the graft interface remain elusive due to the difficulty of locating the exact interface at the ultrastructural level. To date, only studies with interfamily grafts showing enough ultrastructural differences were able to reliably localize the grafting interface at the ultrastructural level under electron microscopy. Thanks to the implementation of correlative light electron microscopy (CLEM) approaches where the grafted partners were tagged with fluorescent proteins of different colors, the graft interface was successfully and reliably targeted. Here, we describe a protocol for CLEM for the model plant Arabidopsis thaliana, which unambiguously targets the graft interface at the ultrastructural level. Moreover, this protocol is compatible with immunolocalization and electron tomography acquisition to achieve a three-dimensional view of the ultrastructural events of interest in plant tissues.

Graphical abstract

Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 μm thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall.