RESUMO
Mucopolysaccharidosis type II (MPS II) is a rare lysosomal storage disease (LSD) involving a genetic error in iduronic acid-2-sulfatase (IDS) metabolism that leads to accumulation of glycosaminoglycans within intracellular lysosomes. The primary treatment for MPS II, enzyme replacement therapy, is not effective for central nervous system (CNS) symptoms, such as intellectual disability, because the drugs do not cross the blood-brain barrier. Recently, autophagy has been associated with LSDs. In this study, we examined the morphologic relationship between neuronal damage and autophagy in IDS knockout mice using antibodies against subunit c of mitochondrial adenosine triphosphate (ATP) synthetase and p62. Immunohistological changes suggesting autophagy, such as vacuolation, were observed in neurons, microglia, and pericytes throughout the CNS, and the numbers increased over postnatal development. Oral administration of chloroquine, which inhibits autophagy, did not suppress damage to microglia and pericytes, but greatly reduced neuronal vacuolation and eliminated neuronal cells with abnormal inclusions. Thus, decreasing autophagy appears to prevent neuronal degeneration. These results suggest that an autophagy modulator could be used in addition to conventional enzyme replacement therapy to preserve the CNS in patients with MPS II.
Assuntos
Autofagia , Mucopolissacaridose II/metabolismo , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Cloroquina/farmacologia , Iduronato Sulfatase/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , Microglia/metabolismo , Microglia/ultraestrutura , ATPases Mitocondriais Próton-Translocadoras/genética , ATPases Mitocondriais Próton-Translocadoras/metabolismo , Mucopolissacaridose II/patologia , Neurônios/efeitos dos fármacos , Neurônios/ultraestrutura , Proteína Sequestossoma-1/genética , Proteína Sequestossoma-1/metabolismoRESUMO
The adhesion of circulating lymphocytes to the surface of vascular endothelial cells is important for their recruitment from blood to secondary lymphoid organs and to inflammatory sites. CD44 is a key adhesion molecule for this interaction and its ligand-binding ability is tightly regulated. Here we show that the hyaluronan-binding ability of CD44 in T cells is upregulated by the depletion of membrane cholesterol with methyl-ß-cyclodextrin (MßCD), which disintegrates lipid rafts, i.e. cholesterol- and sphingolipid-enriched membrane microdomains. Increasing concentrations of MßCD led to a dose-dependent decrease in cellular cholesterol content and to upregulation of hyaluronan binding. Additionally, a cholesterol-binding agent filipin also increased hyaluronan binding. Cholesterol depletion caused CD44 to be dispersed from cholesterol-enriched membrane microdomains. Cholesterol depletion also increased the number of cells undergoing rolling adhesion under physiological flow conditions. Our results suggest that the ligand-binding ability of CD44 is governed by its cholesterol-dependent allocation to membrane microdomains at the cell surface. These findings provide novel insight into the regulation of T cell adhesion under blood flow.
Assuntos
Colesterol/metabolismo , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Linfócitos T/metabolismo , Animais , Adesão Celular/fisiologia , Linhagem Celular , Membrana Celular/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Linfócitos T/citologiaRESUMO
High-throughput immuno-electron microscopy is required to capture the protein-protein interactions realizing physiological functions. Atmospheric scanning electron microscopy (ASEM) allows in situ correlative light and electron microscopy of samples in liquid in an open atmospheric environment. Cells are cultured in a few milliliters of medium directly in the ASEM dish, which can be coated and transferred to an incubator as required. Here, cells were imaged by optical or fluorescence microscopy, and at high resolution by gold-labeled immuno-ASEM, sometimes with additional metal staining. Axonal partitioning of neurons was correlated with specific cytoskeletal structures, including microtubules, using primary-culture neurons from wild type Drosophila, and the involvement of ankyrin in the formation of the intra-axonal segmentation boundary was studied using neurons from an ankyrin-deficient mutant. Rubella virus replication producing anti-double-stranded RNA was captured at the host cell's plasma membrane. Fas receptosome formation was associated with clathrin internalization near the surface of primitive endoderm cells. Positively charged Nanogold clearly revealed the cell outlines of primitive endoderm cells, and the cell division of lactic acid bacteria. Based on these experiments, ASEM promises to allow the study of protein interactions in various complexes in a natural environment of aqueous liquid in the near future.
Assuntos
Animais Geneticamente Modificados , Técnicas Citológicas/métodos , Drosophila/citologia , Microscopia Eletrônica de Varredura/métodos , Microscopia Imunoeletrônica/métodos , Animais , Endoderma/citologia , Lactobacillales/citologia , Lactobacillales/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Cultura Primária de Células , Vírus da Rubéola/fisiologia , Coloração e Rotulagem/métodos , Replicação ViralRESUMO
The living body is composed of innumerable fine and complex structures. Although these structures have been studied in the past, a vast amount of information pertaining to them still remains unknown. When attempting to observe these ultra-structures, the use of electron microscopy (EM) has become indispensable. However, conventional EM settings are limited to a narrow tissue area, which can bias observations. Recently, new trends in EM research have emerged, enabling coverage of far broader, nano-scale fields of view for two-dimensional wide areas and three-dimensional large volumes. Moreover, cutting-edge bioimage informatics conducted via deep learning has accelerated the quantification of complex morphological bioimages. Taken together, these technological and analytical advances have led to the comprehensive acquisition and quantification of cellular morphology, which now arises as a new omics science termed 'morphomics'.
Assuntos
Microscopia Eletrônica , Microscopia Eletrônica/métodos , Humanos , Animais , Imageamento Tridimensional/métodosRESUMO
The adhesion of leukocytes circulating in the blood to vascular endothelium is critical for their trafficking in the vasculature, and CD44 is an important cell surface receptor for rolling adhesion. In this study, we demonstrate the correlative observation of CD44 distribution at the lymphocyte cell surface in liquid by fluorescence optical microscopy and immuno-electron microscopy using an atmospheric scanning electron microscope (ASEM). The ultrastructure of the cell surface was clearly imaged by ASEM using positively charged Nanogold particles. ASEM analysis demonstrated microvilli projections around the cell surface and the localization of CD44 on the microvilli. Treatment of cells with cytochalasin D resulted in a loss of the microvilli projections and concomitantly abrogated CD44-mediated adhesion to its ligand hyaluronan. These results suggest the functional relevance of microvilli in CD44-mediated rolling adhesion under shear flow.
Assuntos
Adesão Celular/fisiologia , Ouro/administração & dosagem , Nanopartículas Metálicas/administração & dosagem , Microvilosidades/ultraestrutura , Animais , Linhagem Celular , Citoesqueleto/metabolismo , Citoesqueleto/fisiologia , Citoesqueleto/ultraestrutura , Receptores de Hialuronatos/metabolismo , Ácido Hialurônico/metabolismo , Linfócitos/metabolismo , Linfócitos/fisiologia , Linfócitos/ultraestrutura , Camundongos , Microscopia Eletrônica de Varredura/métodos , Microvilosidades/metabolismo , Microvilosidades/fisiologiaRESUMO
Axons are thought to be ultrathin membrane cables of a relatively uniform diameter, designed to conduct electrical signals, or action potentials. Here, we demonstrate that unmyelinated axons are not simple cylindrical tubes. Rather, axons have nanoscopic boutons repeatedly along their length interspersed with a thin cable with a diameter of â¼60 nm like pearls-on-a-string. These boutons are only â¼200 nm in diameter and do not have synaptic contacts or a cluster of synaptic vesicles, hence non-synaptic. Our in silico modeling suggests that axon pearling can be explained by the mechanical properties of the membrane including the bending modulus and tension. Consistent with modeling predictions, treatments that disrupt these parameters like hyper- or hypo-tonic solutions, cholesterol removal, and non-muscle myosin II inhibition all alter the degree of axon pearling, suggesting that axon morphology is indeed determined by the membrane mechanics. Intriguingly, neuronal activity modulates the cholesterol level of plasma membrane, leading to shrinkage of axon pearls. Consequently, the conduction velocity of action potentials becomes slower. These data reveal that biophysical forces dictate axon morphology and function and that modulation of membrane mechanics likely underlies plasticity of unmyelinated axons.
RESUMO
In the atmospheric scanning electron microscope (ASEM), an inverted SEM observes the wet sample from beneath an open dish while an optical microscope (OM) observes it from above. The disposable dish with a silicon nitride (SiN) film window can hold a few milliliters of culture medium, and allows various types of cells to be cultured in a stable environment. The use of this system for in situ correlative OM/SEM immuno-microscopy is explored, the efficiency of the required dual-tagged labeling assessed and the imaging capabilities of the ASEM documented. We have visualized the cytoskeletons formed by actin and tubulin, the chaperone PDI that catalyses native disulfide bond formation of proteins in the endoplasmic reticulum (ER) and the calcium sensor STIM1 that is integrated in ER membranes, using established cell lines. In particular, a dynamic string-like gathering of STIM1 was observed on the ER in Jurkat T cells in response to Ca(2+) store depletion. We have also visualized filamentous actin (F-actin) and tubulin in the growth cones of primary-culture neurons as well as in synapses. Further, radially running actin fibers were shown to partly colocalize with concentric bands of the Ca(2+) signaling component Homer1c in the lamellipodia of neuron primary culture growth cones. After synapse formation, neurite configurations were drastically rearranged; a button structure with a fine F-actin frame faces a spine with a different F-actin framework. Based on this work, ASEM correlative microscopy promises to allow the dynamics of various protein complexes to be investigated in the near future.
Assuntos
Microscopia Eletrônica de Varredura/métodos , Microscopia Imunoeletrônica/métodos , Microscopia/métodos , Actinas/metabolismo , Animais , Células COS , Cálcio/metabolismo , Chlorocebus aethiops , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Células HeLa , Humanos , Microscopia Confocal , Tubulina (Proteína)/metabolismoRESUMO
CD44 is a cell surface adhesion molecule for hyaluronan and is implicated in tumor invasion and metastasis. Proteolytic cleavage of CD44 plays a critical role in the migration of tumor cells and is regulated by factors present in the tumor microenvironment, such as hyaluronan oligosaccharides and epidermal growth factor. However, molecular mechanisms underlying the proteolytic cleavage on membranes remain poorly understood. In this study, we demonstrated that cholesterol depletion with methyl-ß-cyclodextrin, which disintegrates membrane lipid rafts, enhances CD44 shedding mediated by a disintegrin and metalloproteinase 10 (ADAM10) and that cholesterol depletion disorders CD44 localization to the lipid raft. We also evaluated the effect of long term cholesterol reduction using a statin agent and demonstrated that statin enhances CD44 shedding and suppresses tumor cell migration on a hyaluronan-coated substrate. Our results indicate that membrane lipid organization regulates CD44 shedding and propose a possible molecular mechanism by which cholesterol reduction might be effective for preventing and treating the progression of malignant tumors.
Assuntos
Movimento Celular , Colesterol/metabolismo , Glioma/metabolismo , Receptores de Hialuronatos/metabolismo , Microdomínios da Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteína ADAM10 , Secretases da Proteína Precursora do Amiloide/genética , Secretases da Proteína Precursora do Amiloide/metabolismo , Linhagem Celular Tumoral , Fator de Crescimento Epidérmico/metabolismo , Fator de Crescimento Epidérmico/farmacologia , Glioma/genética , Glioma/patologia , Humanos , Receptores de Hialuronatos/genética , Ácido Hialurônico/metabolismo , Ácido Hialurônico/farmacologia , Microdomínios da Membrana/genética , Microdomínios da Membrana/patologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Metástase Neoplásica , Proteínas de Neoplasias/genética , beta-Ciclodextrinas/farmacologiaRESUMO
Mycoplasma is a genus of bacterial pathogen that causes disease in vertebrates. In humans, the species Mycoplasma pneumoniae causes 15% or more of community-acquired pneumonia. Because this bacterium is tiny, corresponding in size to a large virus, diagnosis using optical microscopy is not easy. In current methods, chest X-rays are usually the first action, followed by serology, PCR amplification, and/or culture, but all of these are particularly difficult at an early stage of the disease. Using Mycoplasma mobile as a model species, we directly observed mycoplasma in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). This microscope features an open sample dish with a pressure-resistant thin film window in its base, through which the SEM beam scans samples in solution, from below. Because of its 2-3µm-deep scanning capability, it can observe the whole internal structure of mycoplasma cells stained with metal solutions. Characteristic protein localizations were visualized using immuno-labeling. Cells were observed at low concentrations, because suspended cells concentrate in the observable zone by attaching to sialic acid on the silicon nitride (SiN) film surface within minutes. These results suggest the applicability of the ASEM for the study of mycoplasmas as well as for early-stage mycoplasma infection diagnosis.
Assuntos
Microscopia Eletrônica de Varredura/métodos , Mycoplasma/isolamento & purificação , Mycoplasma/ultraestrutura , Proteínas de Bactérias/análise , Proteínas de Bactérias/metabolismo , Imuno-Histoquímica , Mycoplasma/metabolismo , Ácido N-Acetilneuramínico/química , Compostos de Silício/química , SoluçõesRESUMO
X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called "crystallization bottleneck". Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iß in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few µm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals.
Assuntos
Lipase/química , Microscopia Eletrônica de Varredura/métodos , Muramidase/química , Proteínas Pol1 do Complexo de Iniciação de Transcrição/química , Animais , Galinhas , Cristalização , Cristalografia por Raios X , Humanos , Lipase/metabolismo , Muramidase/metabolismo , Proteínas Pol1 do Complexo de Iniciação de Transcrição/metabolismoRESUMO
Three-dimensional (3D) observation of a biological sample using serial-section electron microscopy is widely used. However, organelle segmentation requires a significant amount of manual time. Therefore, several studies have been conducted to improve organelle segmentation's efficiency. One such promising method is 3D deep learning (DL), which is highly accurate. However, the creation of training data for 3D DL still requires manual time and effort. In this study, we developed a highly efficient integrated image segmentation tool that includes stepwise DL with manual correction. The tool has four functions: efficient tracers for annotation, model training/inference for organelle segmentation using a lightweight convolutional neural network, efficient proofreading and model refinement. We applied this tool to increase the training data step by step (stepwise annotation method) to segment the mitochondria in the cells of the cerebral cortex. We found that the stepwise annotation method reduced the manual operation time by one-third compared with the fully manual method, where all the training data were created manually. Moreover, we demonstrated that the F1 score, the metric of segmentation accuracy, was 0.9 by training the 3D DL model with these training data. The stepwise annotation method using this tool and the 3D DL model improved the segmentation efficiency of various organelles.
RESUMO
During autophagy, autophagosomes are formed to engulf cytoplasmic contents. p62/SQSTM-1 is an autophagic adaptor protein that forms p62 bodies. A unique feature of p62 bodies is that they seem to directly associate with membranous structures. We first investigated the co-localization of mKate2-p62 bodies with phospholipids using click chemistry with propargyl-choline. Propargyl-choline-labeled phospholipids were detected inside the mKate2-p62 bodies, suggesting that phospholipids were present inside the bodies. To clarify whether or not p62 bodies come in contact with membranous structures directly, we investigated the ultrastructures of p62 bodies using in-resin correlative light and electron microscopy of the Epon-embedded cells expressing mKate2-p62. Fluorescent-positive p62 bodies were detected as uniformly lightly osmificated structures by electron microscopy. Membranous structures were detected on and inside the p62 bodies. In addition, multimembranous structures with rough endoplasmic reticulum-like structures that resembled autophagosomes directly came in contact with amorphous-shaped p62 bodies. These results suggested that p62 bodies are unique structures that can come in contact with membranous structures directly.
Assuntos
Autofagia , Estruturas da Membrana Celular/metabolismo , Proteína Sequestossoma-1/metabolismo , Autofagossomos/metabolismo , Autofagossomos/ultraestrutura , Estruturas da Membrana Celular/ultraestrutura , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Células HeLa , Humanos , Fosfolipídeos/metabolismo , Proteína Sequestossoma-1/análiseRESUMO
Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry.
Assuntos
Membranas Artificiais , Microscopia Eletrônica de Varredura/métodos , Compostos de Silício/química , Animais , Células COS , Chlorocebus aethiopsRESUMO
Direct observation of subcellular structures and their characterization is essential for understanding their physiological functions. To observe them in open environment, we have developed an inverted scanning electron microscope with a detachable, open-culture dish, capable of 8 nm resolution, and combined with a fluorescence microscope quasi-simultaneously observing the same area from the top. For scanning electron microscopy from the bottom, a silicon nitride film window in the base of the dish maintains a vacuum between electron gun and open sample dish while allowing electrons to pass through. Electrons are backscattered from the sample and captured by a detector under the dish. Cells cultured on the open dish can be externally manipulated under optical microscopy, fixed, and observed using scanning electron microscopy. Once fine structures have been revealed by scanning electron microscopy, their component proteins may be identified by comparison with separately prepared fluorescence-labeled optical microscopic images of the candidate proteins, with their heavy-metal-labeled or stained ASEM images. Furthermore, cell nuclei in a tissue block stained with platinum-blue were successfully observed without thin-sectioning, which suggests the applicability of this inverted scanning electron microscope to cancer diagnosis. This microscope visualizes mesoscopic-scale structures, and is also applicable to non-bioscience fields including polymer chemistry.
Assuntos
Membranas Artificiais , Microscopia Eletrônica de Varredura/instrumentação , Microscopia Eletrônica de Varredura/métodos , Compostos de Silício/química , Animais , Encéfalo/citologia , Encéfalo/ultraestrutura , Células COS , Núcleo Celular/ultraestrutura , Chlorocebus aethiops , Cromossomos/ultraestrutura , Endocitose/fisiologia , Retículo Endoplasmático/ultraestrutura , Carpa Dourada , Microscopia de Fluorescência , Mitose/fisiologia , Neurônios/ultraestrutura , Células PC12 , Polissacarídeos/metabolismo , Ratos , Coloração e Rotulagem/métodos , Sinapses/ultraestruturaAssuntos
Anticorpos/análise , Microscopia Eletrônica de Varredura/métodos , Microscopia Imunoeletrônica/métodos , Animais , Humanos , Imunoensaio/métodos , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Microscopia Eletrônica de Varredura/instrumentação , Microscopia Imunoeletrônica/instrumentação , SoluçõesRESUMO
Segmentation of three-dimensional (3D) electron microscopy (EM) image stacks is an arduous and tedious task. Deep convolutional neural networks (CNNs) work well to automate the segmentation; however, they require a large training dataset, which is a major impediment. In order to solve this issue, especially for sparse segmentation, we used a CNN with a minimal training dataset. We segmented a Cerebellar Purkinje cell from an image stack of a mouse Cerebellum cortex in less than two working days, which is much shorter than that of the conventional method. We concluded that we can reduce the total labor time for the sparse segmentation by reducing the training dataset.
Assuntos
Processamento de Imagem Assistida por Computador/métodos , Microscopia Eletrônica , Redes Neurais de Computação , Algoritmos , Aprendizado de Máquina , Células de Purkinje/ultraestruturaRESUMO
In the dorsal root ganglia (DRG), two types of glial cells (Schwann cells and satellite glial cells) have been identified based on cell morphology and expression of specific markers. In the present study, we observed unknown glial cells that were positive for p75 neurotrophin receptor (p75NTR), and therefore were immunohistochemically and ultrastructurally characterized for the first time. These cells exhibited stronger immunoreactivity against an anti-p75NTR antibody than the DRG neurons (hereafter referred to as p75NTR++ cells). Moreover, these cells covered the glial cells surrounding proximal process of the large-diameter DRG neurons. The proximal process is called "dendro-axon." The p75NTR++ cells were predominantly distributed where the first myelinating Schwann cells appear. The p75NTR++ cells were also positive for the pan-glial cell markers S100, nestin, and Sox10, but negative for fibroblast and macrophage markers. Moreover, they were negative for a satellite glial cell marker, inwardly rectifying potassium channel Kir4.1, as well as a nonmyelinating Schwann cell marker, glial fibrillary acidic protein. In addition, their morphological features were distinct from those of the myelinating Schwann cells. To investigate the three-dimensional ultrastructure of the p75NTR++ cells, we used array tomography combined with correlative light and electron microscopic observation. Three-dimensional ultrastructural observation revealed that the p75NTR++ cells loosely covered glial cells around the dendro-axons with highly ramified processes. Glial cells with these morphological features have not been reported before, indicating that the p75NTR++ glial cells are a new glial cell type in the DRG. Our results will give new insights into cell-cell relationships.
Assuntos
Gânglios Espinais/citologia , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/citologia , Receptores de Fatores de Crescimento/metabolismo , Animais , Gânglios Espinais/metabolismo , Masculino , Neuroglia/metabolismo , Ratos , Ratos WistarRESUMO
Wet specimens are notoriously difficult to image in scanning electron microscopes (SEM) owing to evaporation from the required vacuum of the specimen chamber. Traditionally, this issue has been addressed by increasing the specimen chamber pressure. Unfortunately, observation under high specimen chamber pressure cannot prevent the initial evaporation effects. The wet cover method, where the original surface water is retained (and, therefore, considered wet), provides a way to introduce and subsequently image specimens that are sensitive to evaporation within a SEM, while preventing evaporation-related damage, and to observe interesting specimen-water interactions.
Assuntos
Microscopia Eletrônica de Varredura/métodos , Manejo de Espécimes/métodos , Água , Manejo de Espécimes/instrumentação , VácuoRESUMO
The ability to correlate fluorescence microscopy (FM) and electron microscopy (EM) data obtained on biological (cell and tissue) specimens is essential to bridge the resolution gap between the data obtained by these different imaging techniques. In the past such correlations were limited to either EM navigation in two dimensions to the locations previously highlighted by fluorescence markers, or subsequent high-resolution acquisition of tomographic information using a TEM. We present a novel approach whereby a sample previously investigated by FM is embedded and subjected to sequential mechanical polishing and backscatter imaging by scanning electron microscope. The resulting three dimensional EM tomogram of the sample can be directly correlated to the FM data. © 2017 by John Wiley & Sons, Inc.
Assuntos
Imageamento Tridimensional , Microscopia Eletrônica de Varredura/métodos , Microscopia de Fluorescência/métodos , Células HeLa , Humanos , Rim/ultraestruturaRESUMO
"Array tomography" is a method used to observe the fine structure of cells and tissues in a three-dimensional view. In this method, serial ultrathin sections in the ribbon state (ribbons) are mounted on a solid substrate and observed by scanning electron microscopy (SEM). The method may also be used in conjunction with post-embedding immunocytochemistry. However, it is difficult to mount many serial ribbons on a substrate manually. We developed an inexpensive laboratory-made device that mounts ribbons by pulling a nylon fishing line and lifting the substrate up from the water in a knife boat. Using this device, we succeeded in mounting several ribbons consisting a mean of 205.6 (SD: 37.7) serial ultrathin sections on 1.25 (SD: 0.06) × 1.25 (SD: 0.06)-cm silicon substrates. Furthermore, it was confirmed that our method is suitable for ribbons derived from water-soluble resin blocks. We were also able to stain the specimens by post-embedding immunocytochemistry. Thus, our method is useful in mounting numerus sections on a substrate for array tomography with SEM.