细胞生物学


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现刊
往期刊物
0 Q&A 732 Views Dec 20, 2024

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.

0 Q&A 422 Views Nov 20, 2024

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.

0 Q&A 1055 Views Dec 5, 2022

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.

0 Q&A 5213 Views Sep 20, 2019
A protocol was developed to visualize and analyze the structure of membrane vesicles (MVs) from Gram-negative bacteria. It is now accepted that these micrometric spherical vesicles are commonly produced by cells from all three domains of life, so the protocol could be useful in the study of vesicles produced by eukaryotes and archaea as well as bacteria. The multiplicity of functions performed by MVs, related to cell communication, interaction with the immune system, pathogenesis, and nutrient acquisition, among others, has made MVs a hot topic of research.

Due to their small size (25-300 nm), the observation of MVs requires electron microscopy and is usually performed by transmission electron microscopy (TEM) of negatively stained MVs. Other protocols applied for their visualization include scanning electron microscopy, TEM after fixation and embedding of vesicles, or even atomic force microscopy. In some of these techniques, vesicle structure is altered by drying, while others are time-consuming and most of them can generate artifacts. Cryo-TEM after plunge freezing allows the visualization of samples embedded in a thin film of vitreous ice, which preserves their native cellular structures and provides the highest available resolution for the imaging. This is achieved by very high cooling rates that turn the intrinsic water of cells into vitreous ice, avoiding crystal formation and phase segregation between water and solutes. In addition to other types of characterization, an accurate knowledge of MV structure, which can be obtained by this protocol, is essential for MV application in different fields.
0 Q&A 6391 Views Jul 20, 2019
Multibeam scanning electron microscopy (multiSEM) provides a technical platform for seamless nano-to-mesoscale mapping of cells in human tissues and organs, which is a major new initiative of the U.S. National Institutes of Health. Such cross-length-scale imaging is expected to provide unprecedented understanding of relationships between cellular health and tissue-organ as well as organismal-scale health outcomes. For example, understanding relationships between loss in cell viability and cell network connectivity enables identification of emergent behaviors and prediction of degenerative disease onset, in organs as diverse as bone and brain, at early timepoints, providing a basis for future treatments and prevention. Developed for rapid throughput imaging of minute defects on semiconductor wafers, multiSEM has recently been adapted for imaging of human organs, their constituent tissues, and their respective cellular inhabitants. Through integration of geospatial approaches, statistical and network modelling, advances in computing and the management of immense datasets, as well as recent developments in machine learning that enable the automation of big data analyses, multiSEM and other cross- cutting imaging technologies have the potential to exert a profound impact on elucidation of disease mechanisms, translating to improvements in human health. Here we provide a protocol for acquisition and preparation of sample specimen sizes of diagnostic relevance for human anatomy and physiology. We discuss challenges and opportunities to integrate this approach with multibeam scanning electron microscopy workflows as well as multiple imaging modalities for mapping of organ and tissue structure and function.
0 Q&A 4406 Views Apr 20, 2019
In this paper, we describe a protocol allowing measurement of the mechanical tension of individual axons grown ex vivo from neural tissue explants. This protocol was developed with primary cultures of olfactory epithelium explants from embryonic (E13.5) mice. It includes a detailed description of explant dissection and culture, as well as the main steps of the procedure for axon tension measurement using the previously established Biomembrane Force Probe.
0 Q&A 5780 Views Apr 5, 2019
Plant cell walls consist of different polysaccharides and structural proteins, which form a rigid layer located outside of the plasma membrane. The wall is also a very dynamic cell composite, which is characterized by complex polysaccharide interactions and various modifications during cell development. The visualization of cell wall components in situ is very challenging due to the small size of cell wall composites (nanometer scale), large diversity of the wall polysaccharides and their complex interactions. This protocol describes immunogold labeling of different cell wall epitopes for high-resolution transmission electron microscopy (TEM). It provides a detailed procedure for collection and preparation of plant material, ultra-thin sectioning, specimen labeling and contrasting. An immunolabeling procedure workflow was optimized to obtain high efficiency of carbohydrates labeling for high-resolution TEM. This method was applied to study plant cell wall characteristics in various plant tissues but could also be applied for other cell components in plant and animal tissues.
0 Q&A 5139 Views Nov 20, 2018
The recent development of 3D electron microscopic techniques for cells and tissues has necessitated the development of new methods for the detection of proteins and protein-complexes in situ. The development of new genetic tags, such as the ascorbate peroxidase, APEX, for electron microscopic detection of tagged proteins has expanded the available toolbox and ushered in a new era in biological electron microscopy. Here, we describe methods for combining conditionally-stable nanobodies to fluorescent protein tags with APEX-based detection. These methods are compatible with detection of low levels of expression of fluorescently-tagged proteins and with detection of protein complexes using split GFP-based complementation methods. We describe a simple protocol for applying these methods to the electron microscopic detection of proteins and protein complexes in cultured cells.
0 Q&A 6358 Views Oct 20, 2018
In plants, macroautophagy, here referred as autophagy, is a degradation pathway during which the double-membrane structure named autophagosome engulfs the cargo and then fuses with vacuole for material recycling.

To investigate the process of autophagy, transmission electron microscopy (TEM) was used to monitor the ultrastructure of autophagic structures and identify the cargo during this process due to its high resolution. Compared to other autophagy examination methods including biochemical assays and confocal microscopy, TEM is the only method that indicates the morphology of autophagic structures in nanoscale, which is considered to be one of the best ways to illustrate the morphology of autophagic intermediates and the substrate of autophagy. Here, we describe the autophagy examination assay using TEM in Nicotiana benthamiana leaf cells.
0 Q&A 6011 Views Apr 20, 2018
Positive-stranded (+) RNA viruses are intracellular pathogens in humans, animals and plants. To build viral replicase complexes (VRCs) viruses manipulate lipid flows and reorganize subcellular membranes. Redesigned membranes concentrate viral and host factors and create an environment that facilitates the formation of VRCs within replication organelles. Therefore, efficient virus replication depends on the assembly of specialized membranes where viral macromolecular complexes are turned on and hold a variety of functions. Detailed characterization of viral replication platforms in cells requires sophisticated imaging approaches. Here we present a protocol to visualize the three-dimensional organization of the tombusvirus replicase complex in yeast with MEtal-Tagging Transmission Electron Microscopy (METTEM). This protocol allowed us to image the intracellular distribution of the viral replicase molecules in three-dimensions with METTEM and electron tomography. Our study showed how viral replicase molecules build replication complexes within specialized cell membranes.