Liquid cell transmission electron microscopy (LCTEM) enables imaging of dynamic processes in liquid with high spatial and temporal resolution. The widely used liquid cell (LC) consists of two stacking microchips with a thin wet sample sandwiched between them. The vertically overlapped electron-transparent membrane windows on the microchips provide passage for the electron beam. However, microchips with imprecise dimensions usually cause poor alignment of the windows and difficulty in acquiring high-quality images. In this study, we developed a new and efficient microchip fabrication process for LCTEM with a large viewing area (180 µm × 40 µm) and evaluated the resultant LC. The new positioning reference marks on the surface of the Si wafer dramatically improve the precision of dicing the wafer, making it possible to accurately align the windows on two stacking microchips. The precise alignment led to a liquid thickness of 125.6 nm close to the edge of the viewing area. The performance of our LC was demonstrated by in situ transmission electron microscopy imaging of the dynamic motions of 2-nm Pt particles. This versatile and cost-effective microchip production method can be used to fabricate other types of microchips for in situ electron microscopy.
{"title":"Retraction to: Self-assembly of nickel icosahedrons and truncated octahedral nanocrystals on a SrTiO3 (111) support","authors":"Atif Rasheed","doi":"10.1093/jmicro/dfab013","DOIUrl":"10.1093/jmicro/dfab013","url":null,"abstract":"Liquid cell transmission electron microscopy (LCTEM) enables imaging of dynamic processes in liquid with high spatial and temporal resolution. The widely used liquid cell (LC) consists of two stacking microchips with a thin wet sample sandwiched between them. The vertically overlapped electron-transparent membrane windows on the microchips provide passage for the electron beam. However, microchips with imprecise dimensions usually cause poor alignment of the windows and difficulty in acquiring high-quality images. In this study, we developed a new and efficient microchip fabrication process for LCTEM with a large viewing area (180 µm × 40 µm) and evaluated the resultant LC. The new positioning reference marks on the surface of the Si wafer dramatically improve the precision of dicing the wafer, making it possible to accurately align the windows on two stacking microchips. The precise alignment led to a liquid thickness of 125.6 nm close to the edge of the viewing area. The performance of our LC was demonstrated by in situ transmission electron microscopy imaging of the dynamic motions of 2-nm Pt particles. This versatile and cost-effective microchip production method can be used to fabricate other types of microchips for in situ electron microscopy.","PeriodicalId":18515,"journal":{"name":"Microscopy","volume":null,"pages":null},"PeriodicalIF":1.8,"publicationDate":"2021-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/jmicro/dfab013","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"39293320","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}
Hangjian Ling;Kaushik Sridhar;Sumanth Gollapudi;Jyoti Kumar;Robert S Ohgami
The measurement of the volume of blood cells is important for clinical diagnosis and patient management. While digital holography microscopy has been used to obtain such information, previous off-axis setups usually involve a separated reference beam and are thus not very easy to implement. Here, we use the simple in-line Gabor setup without separation of a reference beam to measure the shape and volume of cells mounted on glass slides. Inherent to the in-line holograms, the reconstructed phase of the object is affected by the virtual image noise, producing errors in the cell volume measurement. We optimized our approach to use a single hologram without phase retrieval, increasing distance between cell and hologram plane to reduce the measurement error of cell volume to less than 6% in some instances. Therefore, the in-line Gabor setup can be a useful and simple tool to obtain volumetric and morphologic cellular information.
{"title":"Measurement of cell volume using in-line digital holography","authors":"Hangjian Ling;Kaushik Sridhar;Sumanth Gollapudi;Jyoti Kumar;Robert S Ohgami","doi":"10.1093/jmicro/dfaa077","DOIUrl":"10.1093/jmicro/dfaa077","url":null,"abstract":"The measurement of the volume of blood cells is important for clinical diagnosis and patient management. While digital holography microscopy has been used to obtain such information, previous off-axis setups usually involve a separated reference beam and are thus not very easy to implement. Here, we use the simple in-line Gabor setup without separation of a reference beam to measure the shape and volume of cells mounted on glass slides. Inherent to the in-line holograms, the reconstructed phase of the object is affected by the virtual image noise, producing errors in the cell volume measurement. We optimized our approach to use a single hologram without phase retrieval, increasing distance between cell and hologram plane to reduce the measurement error of cell volume to less than 6% in some instances. Therefore, the in-line Gabor setup can be a useful and simple tool to obtain volumetric and morphologic cellular information.","PeriodicalId":18515,"journal":{"name":"Microscopy","volume":null,"pages":null},"PeriodicalIF":1.8,"publicationDate":"2021-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/jmicro/dfaa077","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38758964","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}
Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.
{"title":"A photoswitchable fluorescent protein for hours-time-lapse and sub-second-resolved super-resolution imaging","authors":"Tetsuichi Wazawa;Ryohei Noma;Shusaku Uto;Kazunori Sugiura;Takashi Washio;Takeharu Nagai","doi":"10.1093/jmicro/dfab001","DOIUrl":"10.1093/jmicro/dfab001","url":null,"abstract":"Reversibly photoswitchable fluorescent proteins (RSFPs) are a class of fluorescent proteins whose fluorescence can be turned on and off by light irradiation. RSFPs have become essential tools for super-resolution (SR) imaging. Because most SR imaging techniques require high-power-density illumination, mitigating phototoxicity in cells due to intense light irradiation has been a challenge. Although we previously developed an RSFP named Kohinoor to achieve SR imaging with low phototoxicity, the photoproperties were insufficient to move a step further to explore the cellular dynamics by SR imaging. Here, we show an improved version of RSFP, Kohinoor2.0, which is suitable for SR imaging of cellular processes. Kohinoor2.0 shows a 2.6-fold higher fluorescence intensity, 2.5-fold faster chromophore maturation and 1.5-fold faster off-switching than Kohinoor. The analysis of the pH dependence of the visible absorption band revealed that Kohinoor2.0 and Kohinoor were in equilibria among multiple fluorescently bright and dark states, with the mutations introduced into Kohinoor2.0 bringing about a higher stabilization of the fluorescently bright states compared to Kohinoor. Using Kohinoor2.0 with our SR imaging technique, super-resolution polarization demodulation/on-state polarization angle narrowing, we conducted 4-h time-lapse SR imaging of an actin filament network in mammalian cells with a total acquisition time of 480 s without a noticeable indication of phototoxicity. Furthermore, we demonstrated the SR imaging of mitochondria dynamics at a time resolution of 0.5 s, in which the fusion and fission processes were clearly visualized. Thus, Kohinoor2.0 is shown to be an invaluable RSFP for the SR imaging of cellular dynamics.","PeriodicalId":18515,"journal":{"name":"Microscopy","volume":null,"pages":null},"PeriodicalIF":1.8,"publicationDate":"2021-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/jmicro/dfab001","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38768950","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Alveolar bone repair after tooth extraction is essential after oral surgeries. Various grafting materials are used to promote the regeneration of lost alveolar bone. This study analysed the morphological features of the tissue regeneration process using deproteinized bovine bone mineral (DBBM). DBBM was used to densely fill the extraction sockets in beagle dogs. Following resin casting of the vasculature, stereomicroscopy and scanning electron microscopy were used to observe blood vessels and hard tissues in haematoxylin and eosin-stained sections on postoperative days 14, 30 and 90 in conjunction with vascular endothelial growth factor (VEGF) immunostaining to evaluate alveolar bone vascularization. On day 14 post-operation, the DBBM granules tightly filled the extraction sockets, maintained alveolar margin height and formed a scaffold for aiding angiogenesis and new bone formation. On day 30, new bone formation was observed around the DBBM granules. By day 90, bone tissue regeneration progressed in both groups but was more pronounced in the DBBM group. Alveolar margin height was maintained in the DBBM group throughout the study. Furthermore, VEGF expression in the DBBM group was detected around newly formed bone. We conclude that DBBM acts as a suitable scaffold for new bone generation, as well as angiogenesis around healing alveolar bone, and that it has the potential to play a key role in vascularization and bone formation.
{"title":"Application of deproteinized bovine bone mineral as proangiogenic scaffold for alveolar bone formation in beagle dogs","authors":"Keita Ogasawara;Masahiro To;Yu-Hao Liu;Toshimitsu Okudera;Takatsuna Nakamura;Masato Matsuo","doi":"10.1093/jmicro/dfab007","DOIUrl":"10.1093/jmicro/dfab007","url":null,"abstract":"Alveolar bone repair after tooth extraction is essential after oral surgeries. Various grafting materials are used to promote the regeneration of lost alveolar bone. This study analysed the morphological features of the tissue regeneration process using deproteinized bovine bone mineral (DBBM). DBBM was used to densely fill the extraction sockets in beagle dogs. Following resin casting of the vasculature, stereomicroscopy and scanning electron microscopy were used to observe blood vessels and hard tissues in haematoxylin and eosin-stained sections on postoperative days 14, 30 and 90 in conjunction with vascular endothelial growth factor (VEGF) immunostaining to evaluate alveolar bone vascularization. On day 14 post-operation, the DBBM granules tightly filled the extraction sockets, maintained alveolar margin height and formed a scaffold for aiding angiogenesis and new bone formation. On day 30, new bone formation was observed around the DBBM granules. By day 90, bone tissue regeneration progressed in both groups but was more pronounced in the DBBM group. Alveolar margin height was maintained in the DBBM group throughout the study. Furthermore, VEGF expression in the DBBM group was detected around newly formed bone. We conclude that DBBM acts as a suitable scaffold for new bone generation, as well as angiogenesis around healing alveolar bone, and that it has the potential to play a key role in vascularization and bone formation.","PeriodicalId":18515,"journal":{"name":"Microscopy","volume":null,"pages":null},"PeriodicalIF":1.8,"publicationDate":"2021-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/jmicro/dfab007","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25322188","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}
Liquid cell transmission electron microscopy (LCTEM) enables imaging of dynamic processes in liquid with high spatial and temporal resolution. The widely used liquid cell (LC) consists of two stacking microchips with a thin wet sample sandwiched between them. The vertically overlapped electron-transparent membrane windows on the microchips provide passage for the electron beam. However, microchips with imprecise dimensions usually cause poor alignment of the windows and difficulty in acquiring high-quality images. In this study, we developed a new and efficient microchip fabrication process for LCTEM with a large viewing area (180 µm × 40 µm) and evaluated the resultant LC. The new positioning reference marks on the surface of the Si wafer dramatically improve the precision of dicing the wafer, making it possible to accurately align the windows on two stacking microchips. The precise alignment led to a liquid thickness of 125.6 nm close to the edge of the viewing area. The performance of our LC was demonstrated by in situ transmission electron microscopy imaging of the dynamic motions of 2-nm Pt particles. This versatile and cost-effective microchip production method can be used to fabricate other types of microchips for in situ electron microscopy.
{"title":"Fabrication of a liquid cell for in situ transmission electron microscopy","authors":"Xiaoguang Li;Kazutaka Mitsuishi;Masaki Takeguchi","doi":"10.1093/jmicro/dfaa076","DOIUrl":"10.1093/jmicro/dfaa076","url":null,"abstract":"Liquid cell transmission electron microscopy (LCTEM) enables imaging of dynamic processes in liquid with high spatial and temporal resolution. The widely used liquid cell (LC) consists of two stacking microchips with a thin wet sample sandwiched between them. The vertically overlapped electron-transparent membrane windows on the microchips provide passage for the electron beam. However, microchips with imprecise dimensions usually cause poor alignment of the windows and difficulty in acquiring high-quality images. In this study, we developed a new and efficient microchip fabrication process for LCTEM with a large viewing area (180 µm × 40 µm) and evaluated the resultant LC. The new positioning reference marks on the surface of the Si wafer dramatically improve the precision of dicing the wafer, making it possible to accurately align the windows on two stacking microchips. The precise alignment led to a liquid thickness of 125.6 nm close to the edge of the viewing area. The performance of our LC was demonstrated by in situ transmission electron microscopy imaging of the dynamic motions of 2-nm Pt particles. This versatile and cost-effective microchip production method can be used to fabricate other types of microchips for in situ electron microscopy.","PeriodicalId":18515,"journal":{"name":"Microscopy","volume":null,"pages":null},"PeriodicalIF":1.8,"publicationDate":"2021-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/jmicro/dfaa076","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38711357","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}
Nickel nanocrystals have received much attention for their ferromagnetic properties. The crystal properties are strongly dependent on their facets and therefore detailed study of their morphology, facets and orientation is critical for magnetic applications. In this work, equilibrium crystal shapes of self-assembled nickel nanocrystals on the (111) termination of strontium titanate (SrTiO 3 ) at room temperature and under ultra-high vacuum (UHV) conditions have been investigated using scanning tunneling microscope. SrTiO 3 (111) substrate was sputtered (0.5 keV, 2.5 µA, 10 min) and annealed (900°C, 1 h) under UHV conditions. Three different periodicities were observed: 2.21 ± 0.01 nm corresponding to (4 × 4) reconstruction, 3.31 ± 0.02 nm corresponding to (6 × 6) reconstruction and 2.85 ± 0.05 nm, rotated at 30° with respect to (4 × 4) reconstruction, corresponding to (3√3 × 3√3)R30° reconstruction. Nickel (∼1 ml) was deposited using an e-beam evaporator on the substrate preheated to 320°C and the sample was post-annealed multiple times. Nickel took platonic shapes of supported icosahedron comprising of (111) facets and truncated octahedron comprising of (001) and (111) facets. Based on surface energy ratios of truncated octahedrons at equilibrium, the work of adhesion was calculated to be 3.889 ± 0.167 J/m 2 .
{"title":"Self-assembly of nickel icosahedrons and truncated octahedral nanocrystals on a SrTiO3 (111) support","authors":"Atif Rasheed","doi":"10.1093/jmicro/dfaa078","DOIUrl":"10.1093/jmicro/dfaa078","url":null,"abstract":"Nickel nanocrystals have received much attention for their ferromagnetic properties. The crystal properties are strongly dependent on their facets and therefore detailed study of their morphology, facets and orientation is critical for magnetic applications. In this work, equilibrium crystal shapes of self-assembled nickel nanocrystals on the (111) termination of strontium titanate (SrTiO\u0000<inf>3</inf>\u0000) at room temperature and under ultra-high vacuum (UHV) conditions have been investigated using scanning tunneling microscope. SrTiO\u0000<inf>3</inf>\u0000 (111) substrate was sputtered (0.5 keV, 2.5 µA, 10 min) and annealed (900°C, 1 h) under UHV conditions. Three different periodicities were observed: 2.21 ± 0.01 nm corresponding to (4 × 4) reconstruction, 3.31 ± 0.02 nm corresponding to (6 × 6) reconstruction and 2.85 ± 0.05 nm, rotated at 30° with respect to (4 × 4) reconstruction, corresponding to (3√3 × 3√3)R30° reconstruction. Nickel (∼1 ml) was deposited using an e-beam evaporator on the substrate preheated to 320°C and the sample was post-annealed multiple times. Nickel took platonic shapes of supported icosahedron comprising of (111) facets and truncated octahedron comprising of (001) and (111) facets. Based on surface energy ratios of truncated octahedrons at equilibrium, the work of adhesion was calculated to be 3.889 ± 0.167 J/m\u0000<sup>2</sup>\u0000.","PeriodicalId":18515,"journal":{"name":"Microscopy","volume":null,"pages":null},"PeriodicalIF":1.8,"publicationDate":"2021-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/jmicro/dfaa078","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38759348","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}
In transmission electron microscope (TEM), both the amplitude and the phase of electron beam change when electrons traverse a specimen. The amplitude is easily obtained by the square root of the intensity of a TEM image, while the phase affects defocused images. In order to obtain the phase map and verify the theoretical model of the interaction between electron beam and specimen, a lot of simulations have to be performed by researchers. In this work, we have simulated defocus images of a SiC nanowire in TEM with the method of electron optics. Mean inner potential and charge distribution on the nanowire have been considered in the simulation. Besides, due to electron scattering, coherence loss of the electron beam has been introduced. A dynamic process with Bayesian optimization was used in the simulation. With the infocus image as input and by adjusting fitting parameters, the defocus image is determined with a reasonable charge distribution. The calculated defocus images are in a good agreement with the experimental ones. Here, we present a complete solution and verification method for solving nanoscale charge distribution in TEM.
{"title":"Imaging simulation of charged nanowires in TEM with large defocus distance","authors":"Te Shi;Shikai Liu;H Tian;Z J Ding","doi":"10.1093/jmicro/dfab008","DOIUrl":"10.1093/jmicro/dfab008","url":null,"abstract":"In transmission electron microscope (TEM), both the amplitude and the phase of electron beam change when electrons traverse a specimen. The amplitude is easily obtained by the square root of the intensity of a TEM image, while the phase affects defocused images. In order to obtain the phase map and verify the theoretical model of the interaction between electron beam and specimen, a lot of simulations have to be performed by researchers. In this work, we have simulated defocus images of a SiC nanowire in TEM with the method of electron optics. Mean inner potential and charge distribution on the nanowire have been considered in the simulation. Besides, due to electron scattering, coherence loss of the electron beam has been introduced. A dynamic process with Bayesian optimization was used in the simulation. With the infocus image as input and by adjusting fitting parameters, the defocus image is determined with a reasonable charge distribution. The calculated defocus images are in a good agreement with the experimental ones. Here, we present a complete solution and verification method for solving nanoscale charge distribution in TEM.","PeriodicalId":18515,"journal":{"name":"Microscopy","volume":null,"pages":null},"PeriodicalIF":1.8,"publicationDate":"2021-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"25408565","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}
Degradation of the crystalline quality of transmission electron microscopy specimens in silicon prepared with different conditions has been examined using convergent-beam electron diffraction (CBED). The specimens are prepared using focused ion beam (FIB) with different accelerating voltages, Ar-ion milling and crushing method. Symmetry breaking of CBED patterns was quantitatively evaluated by symmetry breaking index S, which has been previously reported. The degradation and inhomogeneity of the FIB specimen were suppressed by decreasing the accelerating voltages of the FIB fabrication in the final process.
{"title":"Evaluation of TEM specimen quality prepared by focused ion beam using symmetry breaking index of convergent-beam electron diffraction","authors":"Daisuke Morikawa;Masaki Ageishi;Kaori Sato;Kenji Tsuda;Masami Terauchi","doi":"10.1093/jmicro/dfab002","DOIUrl":"10.1093/jmicro/dfab002","url":null,"abstract":"Degradation of the crystalline quality of transmission electron microscopy specimens in silicon prepared with different conditions has been examined using convergent-beam electron diffraction (CBED). The specimens are prepared using focused ion beam (FIB) with different accelerating voltages, Ar-ion milling and crushing method. Symmetry breaking of CBED patterns was quantitatively evaluated by symmetry breaking index S, which has been previously reported. The degradation and inhomogeneity of the FIB specimen were suppressed by decreasing the accelerating voltages of the FIB fabrication in the final process.","PeriodicalId":18515,"journal":{"name":"Microscopy","volume":null,"pages":null},"PeriodicalIF":1.8,"publicationDate":"2021-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38756160","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}
Methodology for quantitative evaluation of electron radiation damage and calculation of tolerable electron dose was developed to achieve damage-less scanning electron microscope (SEM) observation of beam-sensitive polymer film. The radiation damage is typically evaluated with visual impressions of SEM images; however, this method may be unreliable because observer's subjectivity may affect the results. Evaluation with quantitative value is crucial to improve reliability. In this study, the radiation damage was evaluated by using normalized correlative coefficient (R NCC ) between an initial frame and latter frames of the multiple SEM images that were taken consecutively. Tolerable dose was obtained by defining a threshold point of R NCC where rapid reduction of R NCC started. A SEM image with less damage and acceptable signal-to-noise ratio was obtained by integrating the images from the initial frame to the tolerable frame.
{"title":"Damage-less observation of polymers by electron dose control in scanning electron microscope","authors":"Yoichiro Hashimoto;Kunji Shigeto;Ryo Komatsuzaki;Tsutomu Saito;Takashi Sekiguchi","doi":"10.1093/jmicro/dfab006","DOIUrl":"10.1093/jmicro/dfab006","url":null,"abstract":"Methodology for quantitative evaluation of electron radiation damage and calculation of tolerable electron dose was developed to achieve damage-less scanning electron microscope (SEM) observation of beam-sensitive polymer film. The radiation damage is typically evaluated with visual impressions of SEM images; however, this method may be unreliable because observer's subjectivity may affect the results. Evaluation with quantitative value is crucial to improve reliability. In this study, the radiation damage was evaluated by using normalized correlative coefficient (R\u0000<inf>NCC</inf>\u0000) between an initial frame and latter frames of the multiple SEM images that were taken consecutively. Tolerable dose was obtained by defining a threshold point of R\u0000<inf>NCC</inf>\u0000 where rapid reduction of R\u0000<inf>NCC</inf>\u0000 started. A SEM image with less damage and acceptable signal-to-noise ratio was obtained by integrating the images from the initial frame to the tolerable frame.","PeriodicalId":18515,"journal":{"name":"Microscopy","volume":null,"pages":null},"PeriodicalIF":1.8,"publicationDate":"2021-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/jmicro/dfab006","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38867149","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}
Correlative light and electron microscopy (CLEM) is an excellent approach for examining the cellular localization of biomolecules. Here, we developed a simple method for CLEM by combining pre-embedding immunohistochemistry with a novel fluorescent probe, namely Fluolid NS Orange, and an embedding resin called ʻDurcupan ™ ʼ. Specimens were embedded in Durcupan ™ or LR White after immunolabeling and post-fixation using glutaraldehyde and osmium tetroxide. Next, ultrathin sections were prepared on a finder grid with navigation markers. The section of the specimen embedded in Durcupan ™ was found to be more stable against electron beam irradiation than specimens embedded in LR White. A fluorescence light microscopy image and a transmission electron microscopy (TEM) image, at wide-field, and low magnification, were independently obtained with the same ultrathin section. Using the three corners between finder grid bars as landmarks, fluorescence light microscopy images were superimposed with wide-field, low-magnification TEM images to identify the region of interest, which was subsequently enlarged to ascertain cellular structures localized beneath fluorescent signals. However, the enlarged TEM images appeared blurred, and fluorescence signals had a hazy appearance. To resolve this, the enlarged TEM images were replaced by high-resolution TEM images focused directly on the region of interest, thereby facilitating the collection of high-resolution CLEM images. The simple sample processing method for CLEM using osmium-resistant Fluolid NS Orange and electron beam damage-resistant Durcupan™ allowed the determination of the precise localization of fluorescence signals at subcellular levels.
{"title":"A simple preparation method for CLEM using pre-embedding immunohistochemistry with a novel fluorescent probe and stable embedding resin","authors":"Takaaki Kanemaru;Teruyoshi Kondo;Kei-ichiro Nakamura;Hiroyuki Morimoto;Kentaro Nishi;Shin-ichiro Isobe","doi":"10.1093/jmicro/dfab005","DOIUrl":"10.1093/jmicro/dfab005","url":null,"abstract":"Correlative light and electron microscopy (CLEM) is an excellent approach for examining the cellular localization of biomolecules. Here, we developed a simple method for CLEM by combining pre-embedding immunohistochemistry with a novel fluorescent probe, namely Fluolid NS Orange, and an embedding resin called ʻDurcupan\u0000<sup>™</sup>\u0000ʼ. Specimens were embedded in Durcupan\u0000<sup>™</sup>\u0000 or LR White after immunolabeling and post-fixation using glutaraldehyde and osmium tetroxide. Next, ultrathin sections were prepared on a finder grid with navigation markers. The section of the specimen embedded in Durcupan\u0000<sup>™</sup>\u0000 was found to be more stable against electron beam irradiation than specimens embedded in LR White. A fluorescence light microscopy image and a transmission electron microscopy (TEM) image, at wide-field, and low magnification, were independently obtained with the same ultrathin section. Using the three corners between finder grid bars as landmarks, fluorescence light microscopy images were superimposed with wide-field, low-magnification TEM images to identify the region of interest, which was subsequently enlarged to ascertain cellular structures localized beneath fluorescent signals. However, the enlarged TEM images appeared blurred, and fluorescence signals had a hazy appearance. To resolve this, the enlarged TEM images were replaced by high-resolution TEM images focused directly on the region of interest, thereby facilitating the collection of high-resolution CLEM images. The simple sample processing method for CLEM using osmium-resistant Fluolid NS Orange and electron beam damage-resistant Durcupan™ allowed the determination of the precise localization of fluorescence signals at subcellular levels.","PeriodicalId":18515,"journal":{"name":"Microscopy","volume":null,"pages":null},"PeriodicalIF":1.8,"publicationDate":"2021-08-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://sci-hub-pdf.com/10.1093/jmicro/dfab005","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"38866672","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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