Autophagy is involved in various fungal morphogenetic processes. However, there are limited reports regarding the role of autophagy in mushroom fruiting body formation. The purpose of this study was to reveal the autophagy-related structures in mushroom-forming fungi. The edible mushroom Pleurotus ostreatus was used in this study. Transmission electron microscopy revealed double-membrane bounded structures containing cytoplasmic components in the fruiting bodies of this fungus. Some of these double-membrane structures were observed to interact with the vacuoles. Additionally, curved flat cisternae of various lengths were detected in the cytoplasm. The shape, size, and thickness of the limiting membrane of the double-membrane structures and the flat cisternae corresponded well with those of the autophagosomes and the isolation membranes, respectively. Regarding autophagosome formation, a membrane-bound specific zone was detected near the isolation membrane, which appeared to expand along the novel membrane. This is the first detailed report showing autophagy-related structures in P. ostreatus and provides a possible model for autophagosome formation in these filamentous fungi. Mini-abstract Autophagy is involved in fungal morphogenetic processes. The fruiting bodies of edible mushroom Pleurotus ostreatus was observed under a TEM. The present study showed autophagy-related structures in this fungus and provides a possible model for autophagosome formation in filamentous fungi.
{"title":"Detection of Autophagy-Related Structures in Fruiting Bodies of Edible Mushroom, Pleurotus ostreatus.","authors":"Yuma Ozaki, T. Aimi, N. Shimomura","doi":"10.1093/jmicro/dfac020","DOIUrl":"https://doi.org/10.1093/jmicro/dfac020","url":null,"abstract":"Autophagy is involved in various fungal morphogenetic processes. However, there are limited reports regarding the role of autophagy in mushroom fruiting body formation. The purpose of this study was to reveal the autophagy-related structures in mushroom-forming fungi. The edible mushroom Pleurotus ostreatus was used in this study. Transmission electron microscopy revealed double-membrane bounded structures containing cytoplasmic components in the fruiting bodies of this fungus. Some of these double-membrane structures were observed to interact with the vacuoles. Additionally, curved flat cisternae of various lengths were detected in the cytoplasm. The shape, size, and thickness of the limiting membrane of the double-membrane structures and the flat cisternae corresponded well with those of the autophagosomes and the isolation membranes, respectively. Regarding autophagosome formation, a membrane-bound specific zone was detected near the isolation membrane, which appeared to expand along the novel membrane. This is the first detailed report showing autophagy-related structures in P. ostreatus and provides a possible model for autophagosome formation in these filamentous fungi. Mini-abstract Autophagy is involved in fungal morphogenetic processes. The fruiting bodies of edible mushroom Pleurotus ostreatus was observed under a TEM. The present study showed autophagy-related structures in this fungus and provides a possible model for autophagosome formation in filamentous fungi.","PeriodicalId":48655,"journal":{"name":"Microscopy","volume":" ","pages":""},"PeriodicalIF":1.8,"publicationDate":"2022-04-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44591942","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Abstract A small number of oncogenic mutated cells sporadically arise within the epithelial monolayer. Newly emerging Ras- or Src-transformed epithelial cells are often apically eliminated during competitive interactions between normal and transformed cells. Our recent electron microscopy (EM) analyses revealed that characteristic finger-like membrane protrusions are formed at the interface between normal and RasV12-transformed cells via the cdc42–formin-binding protein 17 (FBP17) pathway, potentially playing a positive role in intercellular recognition during apical extrusion. However, the spatial distribution and ultrastructural characteristics of finger-like protrusions remain unknown. In this study, we performed both X–Y and X–Z EM analyses of finger-like protrusions during the apical extrusion of RasV12-transformed cells. Quantification of the distribution and widths of the protrusions showed comparable results between the X–Y and X–Z sections. Finger-like protrusions were observed throughout the cell boundary between normal and RasV12 cells, except for apicalmost tight junctions. In addition, a non-cell-autonomous reduction in protrusion widths was observed between RasV12 cells and surrounding normal cells under the mix culture condition. In the finger-like protrusions, intercellular adhesions via thin electron-dense plaques were observed, implying that immature and transient forms of desmosomes, adherens junctions or unknown weak adhesions were distributed. Interestingly, unlike RasV12-transformed cells, Src-transformed cells form fewer evident protrusions, and FBP17 in Src cells is dispensable for apical extrusion. Collectively, these results suggest that the dynamic reorganization of intercellular adhesions via finger-like protrusions may positively control cell competition between normal and RasV12-transformed cells. Furthermore, our data indicate a cell context–dependent diversity in the modes of apical extrusion.
{"title":"Ultrastructural characteristics of finger-like membrane protrusions in cell competition","authors":"Tomoko Kamasaki, Ryota Uehara, Yasuyuki Fujita","doi":"10.1093/jmicro/dfac017","DOIUrl":"https://doi.org/10.1093/jmicro/dfac017","url":null,"abstract":"Abstract A small number of oncogenic mutated cells sporadically arise within the epithelial monolayer. Newly emerging Ras- or Src-transformed epithelial cells are often apically eliminated during competitive interactions between normal and transformed cells. Our recent electron microscopy (EM) analyses revealed that characteristic finger-like membrane protrusions are formed at the interface between normal and RasV12-transformed cells via the cdc42–formin-binding protein 17 (FBP17) pathway, potentially playing a positive role in intercellular recognition during apical extrusion. However, the spatial distribution and ultrastructural characteristics of finger-like protrusions remain unknown. In this study, we performed both X–Y and X–Z EM analyses of finger-like protrusions during the apical extrusion of RasV12-transformed cells. Quantification of the distribution and widths of the protrusions showed comparable results between the X–Y and X–Z sections. Finger-like protrusions were observed throughout the cell boundary between normal and RasV12 cells, except for apicalmost tight junctions. In addition, a non-cell-autonomous reduction in protrusion widths was observed between RasV12 cells and surrounding normal cells under the mix culture condition. In the finger-like protrusions, intercellular adhesions via thin electron-dense plaques were observed, implying that immature and transient forms of desmosomes, adherens junctions or unknown weak adhesions were distributed. Interestingly, unlike RasV12-transformed cells, Src-transformed cells form fewer evident protrusions, and FBP17 in Src cells is dispensable for apical extrusion. Collectively, these results suggest that the dynamic reorganization of intercellular adhesions via finger-like protrusions may positively control cell competition between normal and RasV12-transformed cells. Furthermore, our data indicate a cell context–dependent diversity in the modes of apical extrusion.","PeriodicalId":48655,"journal":{"name":"Microscopy","volume":"71 1","pages":"195 - 205"},"PeriodicalIF":1.8,"publicationDate":"2022-04-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46296015","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Yuji Kanazawa, M. Nagano, S. Koinuma, S. Sugiyo, Y. Shigeyoshi
Abstract We investigated the effect of aging on the basement membrane (BM) during postinjury muscle recovery. Using a rat model, we found that aging delayed muscle fiber and BM recovery. In addition, expression of BM-related factors peaked 7 days after muscle injury among both young and older rats. Peak expression of collagen IV synthetic factors decreased with age, whereas expression of the degradative factor was unaffected by age. These results suggest that age-related delays in postinjury muscle fiber and BM recovery may be related to the suppression of collagen IV synthetic factors.
{"title":"Effects of aging on basement membrane of tibialis anterior muscle during recovery following muscle injury in rats","authors":"Yuji Kanazawa, M. Nagano, S. Koinuma, S. Sugiyo, Y. Shigeyoshi","doi":"10.1093/jmicro/dfac016","DOIUrl":"https://doi.org/10.1093/jmicro/dfac016","url":null,"abstract":"Abstract We investigated the effect of aging on the basement membrane (BM) during postinjury muscle recovery. Using a rat model, we found that aging delayed muscle fiber and BM recovery. In addition, expression of BM-related factors peaked 7 days after muscle injury among both young and older rats. Peak expression of collagen IV synthetic factors decreased with age, whereas expression of the degradative factor was unaffected by age. These results suggest that age-related delays in postinjury muscle fiber and BM recovery may be related to the suppression of collagen IV synthetic factors.","PeriodicalId":48655,"journal":{"name":"Microscopy","volume":"71 1","pages":"245 - 248"},"PeriodicalIF":1.8,"publicationDate":"2022-03-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60920873","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Ultra-thin silicon nitride (SiN) membranes are critical in microfabrication-based liquid cells (LCs) for transmission electron microscopy. This study used a homemade LC with a 50-nm SiN membrane to study the dynamics of 2.58-nm platinum (Pt) nanoparticles (NPs) in approximately 200-nm deep water. When a strong beam with electron flux ranging from 2.5 × 103 to 1.4 ×106 e-/(nm2·s) was applied to resolve the NPs, the beam caused NP aggregation and even drilled a hole on the top membrane. The hole drilling was prevented by coating a 1-4-nm-thick amorphous carbon layer on both sides of the membrane. The NP aggregation rate also decreased with increasing carbon thickness. After overcoming the aforementioned issues, lattice fringes of the Pt NPs were visible when the NPs were attached to the membrane of the 4-nm-carbon-coated LC containing a thin liquid layer. The effects of the electron beam and carbon on the LC and Pt NPs were investigated and discussed. This work provides a reference for LC-TEM research using strong electron beams.
{"title":"Effect of Amorphous Carbon Coating on the Performance of Liquid Phase Transmission Electron Microscopy (LP-TEM) and the Dynamics of Enclosed Pt Nano-Colloids.","authors":"Xiaoguang Li, K. Mitsuishi, M. Takeguchi","doi":"10.1093/jmicro/dfac012","DOIUrl":"https://doi.org/10.1093/jmicro/dfac012","url":null,"abstract":"Ultra-thin silicon nitride (SiN) membranes are critical in microfabrication-based liquid cells (LCs) for transmission electron microscopy. This study used a homemade LC with a 50-nm SiN membrane to study the dynamics of 2.58-nm platinum (Pt) nanoparticles (NPs) in approximately 200-nm deep water. When a strong beam with electron flux ranging from 2.5 × 103 to 1.4 ×106 e-/(nm2·s) was applied to resolve the NPs, the beam caused NP aggregation and even drilled a hole on the top membrane. The hole drilling was prevented by coating a 1-4-nm-thick amorphous carbon layer on both sides of the membrane. The NP aggregation rate also decreased with increasing carbon thickness. After overcoming the aforementioned issues, lattice fringes of the Pt NPs were visible when the NPs were attached to the membrane of the 4-nm-carbon-coated LC containing a thin liquid layer. The effects of the electron beam and carbon on the LC and Pt NPs were investigated and discussed. This work provides a reference for LC-TEM research using strong electron beams.","PeriodicalId":48655,"journal":{"name":"Microscopy","volume":" ","pages":""},"PeriodicalIF":1.8,"publicationDate":"2022-03-11","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"46772698","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
The assembly of misfolded proteins into amyloid fibrils is associated with amyloidosis, including neurodegenerative diseases, such as Alzheimer's, Parkinson's, and prion diseases. The self-propagation of amyloid fibrils is widely observed in the aggregation pathways of numerous amyloidogenic proteins. This propensity with plasticity in primary nucleation allows amyloid fibril polymorphism, which is correlated with the pathology/phenotypes of patients. Because the interference with the nucleation and replication processes of amyloid fibrils can alter the amyloid structure and the outcome of the disease, these processes can be a target for developing clinical drugs. Single-molecule observation of amyloid fibril replication can be an experimental system to provide the kinetic parameters for simulation studies and confirm the effect of clinical drugs. Here, we review single-molecule observation of the amyloid fibril replication process using fluorescence microscopy and time-lapse atomic force microscopy, including high-speed atomic force microscopy. We discussed the amyloid fibril replication process and combined single-molecule observation results with molecular dynamics simulations. Mini Abstract Structural dynamics in amyloid aggregation is related with various Alzheimer's and Parkinson's disease symptoms. Single-molecule observation using high-speed atomic force microscopy can directly visualize the structural dynamics of individual amyloid aggregate assemblies. Here, we review historical and recent studies of single-molecule observation of amyloid aggregation with supportive molecular dynamics simulation.
{"title":"Single-molecule Observation of Self-Propagating Amyloid Fibrils.","authors":"T. Watanabe-Nakayama, K. Ono","doi":"10.1093/jmicro/dfac011","DOIUrl":"https://doi.org/10.1093/jmicro/dfac011","url":null,"abstract":"The assembly of misfolded proteins into amyloid fibrils is associated with amyloidosis, including neurodegenerative diseases, such as Alzheimer's, Parkinson's, and prion diseases. The self-propagation of amyloid fibrils is widely observed in the aggregation pathways of numerous amyloidogenic proteins. This propensity with plasticity in primary nucleation allows amyloid fibril polymorphism, which is correlated with the pathology/phenotypes of patients. Because the interference with the nucleation and replication processes of amyloid fibrils can alter the amyloid structure and the outcome of the disease, these processes can be a target for developing clinical drugs. Single-molecule observation of amyloid fibril replication can be an experimental system to provide the kinetic parameters for simulation studies and confirm the effect of clinical drugs. Here, we review single-molecule observation of the amyloid fibril replication process using fluorescence microscopy and time-lapse atomic force microscopy, including high-speed atomic force microscopy. We discussed the amyloid fibril replication process and combined single-molecule observation results with molecular dynamics simulations. Mini Abstract Structural dynamics in amyloid aggregation is related with various Alzheimer's and Parkinson's disease symptoms. Single-molecule observation using high-speed atomic force microscopy can directly visualize the structural dynamics of individual amyloid aggregate assemblies. Here, we review historical and recent studies of single-molecule observation of amyloid aggregation with supportive molecular dynamics simulation.","PeriodicalId":48655,"journal":{"name":"Microscopy","volume":"1 1","pages":""},"PeriodicalIF":1.8,"publicationDate":"2022-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41614649","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Electron microscopy of biological materials such as bacteria allows multifaceted analysis to understand their structure and function with high resolution, which is difficult to achieve with optical microscopy. However, the samples are damaged or broken by electron beam irradiation and by the vacuum environment. Here, we observed bacteria in a suspension encapsulated in a graphene sandwich that prevents electron beam damage without the need for fixation. Specifically, we demonstrated in situ scanning electron microscopy observation of Escherichia coli in a graphene sandwich containing a perforated membrane as a spacer, encapsulating non-immobilized E. coli between the graphene layers. However, E. coli activity, such as division, was not observed, although the irradiated cells grew slightly when resuspended under optimal culture conditions. Our findings suggest that the graphene sandwich methodology enables the observation of wet E. coli cells by electron microscopy but requires refinement to allow the live imaging of biological materials.
{"title":"Scanning electron microscopy of Escherichia coli encapsulated in a spacerized graphene sandwich.","authors":"Yuki Sasaki, S. Hirayama, R. Nakao","doi":"10.1093/jmicro/dfac010","DOIUrl":"https://doi.org/10.1093/jmicro/dfac010","url":null,"abstract":"Electron microscopy of biological materials such as bacteria allows multifaceted analysis to understand their structure and function with high resolution, which is difficult to achieve with optical microscopy. However, the samples are damaged or broken by electron beam irradiation and by the vacuum environment. Here, we observed bacteria in a suspension encapsulated in a graphene sandwich that prevents electron beam damage without the need for fixation. Specifically, we demonstrated in situ scanning electron microscopy observation of Escherichia coli in a graphene sandwich containing a perforated membrane as a spacer, encapsulating non-immobilized E. coli between the graphene layers. However, E. coli activity, such as division, was not observed, although the irradiated cells grew slightly when resuspended under optimal culture conditions. Our findings suggest that the graphene sandwich methodology enables the observation of wet E. coli cells by electron microscopy but requires refinement to allow the live imaging of biological materials.","PeriodicalId":48655,"journal":{"name":"Microscopy","volume":" ","pages":""},"PeriodicalIF":1.8,"publicationDate":"2022-02-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"43821219","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Nowadays, sub-50 meV atom-wide electron probes are routinely produced for electron energy loss spectroscopy in transmission electron microscopes due to monochromator technology advances. We review how gradual improvements in energy resolution enabled the study of very low-energy excitations such as lattice phonons, molecular vibrations, infrared plasmons and strongly coupled hybrid modes in nanomaterials. Starting with the theoretical framework needed to treat inelastic electron scattering from phonons in solids, we illustrate contributions in detecting optical surface phonons in photonic structures. We discuss phonon mapping capabilities in real and reciprocal space, and the localized phonon response near nano-/atomic-scale structural features. We also survey the progress of aloof spectroscopy in studying vibrations in organic materials and applications in measuring local temperature and photonic density of states in single nanostructures using phonon scattering. We then turn towards studies on infrared plasmons in metals and semiconductors. Spectroscopy analyses now extend towards probing extremely complex broadband platforms, the effects of defects and nanogaps, and some far-reaching investigations towards uncovering plasmon lifetime and 3D photonic density of states. In doped semiconductors, we review research on the use of the electron probe to correlate local doping concentration and atomic-scale defects with the plasmonic response. Finally, we discuss advances in studying strong coupling phenomena in plasmon-exciton and plasmon-phonon systems. Overall, the wealth of information gained extends our knowledge about nanomaterial properties and elementary excitations, illustrating the powerful capabilities of high-energy resolution scanning transmission electron microscopy-electron energy loss spectrometry.
{"title":"Advances in ultrahigh-energy resolution EELS: phonons, infrared plasmons and strongly coupled modes.","authors":"M. Lagos, I. Bicket, S. Mousavi M, G. Botton","doi":"10.1093/jmicro/dfab050","DOIUrl":"https://doi.org/10.1093/jmicro/dfab050","url":null,"abstract":"Nowadays, sub-50 meV atom-wide electron probes are routinely produced for electron energy loss spectroscopy in transmission electron microscopes due to monochromator technology advances. We review how gradual improvements in energy resolution enabled the study of very low-energy excitations such as lattice phonons, molecular vibrations, infrared plasmons and strongly coupled hybrid modes in nanomaterials. Starting with the theoretical framework needed to treat inelastic electron scattering from phonons in solids, we illustrate contributions in detecting optical surface phonons in photonic structures. We discuss phonon mapping capabilities in real and reciprocal space, and the localized phonon response near nano-/atomic-scale structural features. We also survey the progress of aloof spectroscopy in studying vibrations in organic materials and applications in measuring local temperature and photonic density of states in single nanostructures using phonon scattering. We then turn towards studies on infrared plasmons in metals and semiconductors. Spectroscopy analyses now extend towards probing extremely complex broadband platforms, the effects of defects and nanogaps, and some far-reaching investigations towards uncovering plasmon lifetime and 3D photonic density of states. In doped semiconductors, we review research on the use of the electron probe to correlate local doping concentration and atomic-scale defects with the plasmonic response. Finally, we discuss advances in studying strong coupling phenomena in plasmon-exciton and plasmon-phonon systems. Overall, the wealth of information gained extends our knowledge about nanomaterial properties and elementary excitations, illustrating the powerful capabilities of high-energy resolution scanning transmission electron microscopy-electron energy loss spectrometry.","PeriodicalId":48655,"journal":{"name":"Microscopy","volume":"71 Supplement_1 1","pages":"i174-i199"},"PeriodicalIF":1.8,"publicationDate":"2022-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"44622865","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
H. Terashima, Keiichiro Minatohara, Hisato Maruoka, S. Okabe
Recent advances in human genetics identified genetic variants involved in causing autism spectrum disorders (ASDs). Mouse models that mimic mutations found in patients with ASD exhibit behavioral phenotypes consistent with ASD symptoms. These mouse models suggest critical biological factors of ASD etiology. Another important implication of ASD genetics is the enrichment of ASD risk genes in molecules involved in developing synapses and regulating neural circuit function. Sophisticated in vivo imaging technologies applied to ASD mouse models identify common synaptic impairments in the neocortex, with genetic-mutation-specific defects in local neural circuits. In this article, we review synapse- and circuit-level phenotypes identified by in vivo two-photon imaging in multiple mouse models of ASD and discuss the contributions of altered synapse properties and neural circuit activity to ASD pathogenesis.
{"title":"Imaging neural circuit pathology of autism spectrum disorders: autism-associated genes, animal models and the application of in vivo two-photon imaging.","authors":"H. Terashima, Keiichiro Minatohara, Hisato Maruoka, S. Okabe","doi":"10.1093/jmicro/dfab039","DOIUrl":"https://doi.org/10.1093/jmicro/dfab039","url":null,"abstract":"Recent advances in human genetics identified genetic variants involved in causing autism spectrum disorders (ASDs). Mouse models that mimic mutations found in patients with ASD exhibit behavioral phenotypes consistent with ASD symptoms. These mouse models suggest critical biological factors of ASD etiology. Another important implication of ASD genetics is the enrichment of ASD risk genes in molecules involved in developing synapses and regulating neural circuit function. Sophisticated in vivo imaging technologies applied to ASD mouse models identify common synaptic impairments in the neocortex, with genetic-mutation-specific defects in local neural circuits. In this article, we review synapse- and circuit-level phenotypes identified by in vivo two-photon imaging in multiple mouse models of ASD and discuss the contributions of altered synapse properties and neural circuit activity to ASD pathogenesis.","PeriodicalId":48655,"journal":{"name":"Microscopy","volume":"71 Supplement_1 1","pages":"i81-i99"},"PeriodicalIF":1.8,"publicationDate":"2022-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"45970709","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Genetically encoded tags have introduced extensive lines of application from purification of tagged proteins to their visualization at the single molecular, cellular, histological and whole-body levels. Combined with other rapidly developing technologies such as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, proteomics, super-resolution microscopy and proximity labeling, a large variety of genetically encoded tags have been developed in the last two decades. In this review, I focus on the current status of tag development for electron microscopic (EM) visualization of proteins with metal particle labeling. Compared with conventional immunoelectron microscopy using gold particles, tag-mediated metal particle labeling has several advantages that could potentially improve the sensitivity, spatial and temporal resolution, and applicability to a wide range of proteins of interest (POIs). It may enable researchers to detect single molecules in situ, allowing the quantitative measurement of absolute numbers and exact localization patterns of POI in the ultrastructural context. Thus, genetically encoded tags for EM could revolutionize the field as green fluorescence protein did for light microscopy, although we still have many challenges to overcome before reaching this goal.
{"title":"Electron microscopic visualization of single molecules by tag-mediated metal particle labeling.","authors":"R. Shigemoto","doi":"10.1093/jmicro/dfab048","DOIUrl":"https://doi.org/10.1093/jmicro/dfab048","url":null,"abstract":"Genetically encoded tags have introduced extensive lines of application from purification of tagged proteins to their visualization at the single molecular, cellular, histological and whole-body levels. Combined with other rapidly developing technologies such as clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system, proteomics, super-resolution microscopy and proximity labeling, a large variety of genetically encoded tags have been developed in the last two decades. In this review, I focus on the current status of tag development for electron microscopic (EM) visualization of proteins with metal particle labeling. Compared with conventional immunoelectron microscopy using gold particles, tag-mediated metal particle labeling has several advantages that could potentially improve the sensitivity, spatial and temporal resolution, and applicability to a wide range of proteins of interest (POIs). It may enable researchers to detect single molecules in situ, allowing the quantitative measurement of absolute numbers and exact localization patterns of POI in the ultrastructural context. Thus, genetically encoded tags for EM could revolutionize the field as green fluorescence protein did for light microscopy, although we still have many challenges to overcome before reaching this goal.","PeriodicalId":48655,"journal":{"name":"Microscopy","volume":"71 Supplement_1 1","pages":"i72-i80"},"PeriodicalIF":1.8,"publicationDate":"2022-02-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"60920361","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
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