Spatial Resolution in Scanning Electron Microscopy and Scanning Transmission Electron Microscopy Without a Specimen Vacuum Chamber.

PubMed

Nguyen, Kayla X; Holtz, Megan E; Richmond-Decker, Justin; Muller, David A

2016-08-01

A long-standing goal of electron microscopy has been the high-resolution characterization of specimens in their native environment. However, electron optics require high vacuum to maintain an unscattered and focused probe, a challenge for specimens requiring atmospheric or liquid environments. Here, we use an electron-transparent window at the base of a scanning electron microscope's objective lens to separate column vacuum from the specimen, enabling imaging under ambient conditions, without a specimen vacuum chamber. We demonstrate in-air imaging of specimens at nanoscale resolution using backscattered scanning electron microscopy (airSEM) and scanning transmission electron microscopy. We explore resolution and contrast using Monte Carlo simulations and analytical models. We find that nanometer-scale resolution can be obtained at gas path lengths up to 400 μm, although contrast drops with increasing gas path length. As the electron-transparent window scatters considerably more than gas at our operating conditions, we observe that the densities and thicknesses of the electron-transparent window are the dominant limiting factors for image contrast at lower operating voltages. By enabling a variety of detector configurations, the airSEM is applicable to a wide range of environmental experiments including the imaging of hydrated biological specimens and in situ chemical and electrochemical processes.

Spatial Resolution in Scanning Electron Microscopy and Scanning Transmission Electron Microscopy Without a Specimen Vacuum Chamber

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguyen, Kayla X.; Holtz, Megan E.; Richmond-Decker, Justin

2016-07-25

Abstract A long-standing goal of electron microscopy has been the high-resolution characterization of specimens in their native environment. However, electron optics require high vacuum to maintain an unscattered and focused probe, a challenge for specimens requiring atmospheric or liquid environments. Here, we use an electron-transparent window at the base of a scanning electron microscope’s objective lens to separate column vacuum from the specimen, enabling imaging under ambient conditions, without a specimen vacuum chamber. We demonstrate in-air imaging of specimens at nanoscale resolution using backscattered scanning electron microscopy (airSEM) and scanning transmission electron microscopy. We explore resolution and contrast using Montemore » Carlo simulations and analytical models. We find that nanometer-scale resolution can be obtained at gas path lengths up to 400μm, although contrast drops with increasing gas path length. As the electron-transparent window scatters considerably more than gas at our operating conditions, we observe that the densities and thicknesses of the electron-transparent window are the dominant limiting factors for image contrast at lower operating voltages. By enabling a variety of detector configurations, the airSEM is applicable to a wide range of environmental experiments including the imaging of hydrated biological specimens andin situchemical and electrochemical processes.« less

Electron Diffraction Using Transmission Electron Microscopy

PubMed Central

Bendersky, Leonid A.; Gayle, Frank W.

2001-01-01

Electron diffraction via the transmission electron microscope is a powerful method for characterizing the structure of materials, including perfect crystals and defect structures. The advantages of electron diffraction over other methods, e.g., x-ray or neutron, arise from the extremely short wavelength (≈2 pm), the strong atomic scattering, and the ability to examine tiny volumes of matter (≈10 nm3). The NIST Materials Science and Engineering Laboratory has a history of discovery and characterization of new structures through electron diffraction, alone or in combination with other diffraction methods. This paper provides a survey of some of this work enabled through electron microscopy. PMID:27500060

Consecutive light microscopy, scanning-transmission electron microscopy and transmission electron microscopy of traumatic human brain oedema and ischaemic brain damage.

PubMed

Castejon, O J; Castejon, H V; Diaz, M; Castellano, A

2001-10-01

Cortical biopsies of 11 patients with traumatic brain oedema were consecutively studied by light microscopy (LM) using thick plastic sections, scanning-transmission electron microscopy ((S)TEM) using semithin plastic sections and transmission electron microscopy (TEM) using ultrathin sections. Samples were glutaraldehyde-osmium fixed and embedded in Araldite or Epon. Thick sections were stained with toluidine-blue for light microscopy. Semithin sections were examined unstained and uncoated for (S)TEM. Ultrathin sections were stained with uranyl and lead. Perivascular haemorrhages and perivascular extravasation of proteinaceous oedema fluid were observed in both moderate and severe oedema. Ischaemic pyramidal and non-pyramidal nerve cells appeared shrunken, electron dense and with enlargement of intracytoplasmic membrane compartment. Notably swollen astrocytes were observed in all samples examined. Glycogen-rich and glycogen-depleted astrocytes were identified in anoxic-ischaemic regions. Dark and hydropic satellite, interfascicular and perivascular oligodendrocytes were also found. The status spongiosus of severely oedematous brain parenchyma observed by LM and (S)TEM was correlated with the enlarged extracellular space and disrupted neuropil observed by TEM. The (S)TEM is recommended as a suitable technique for studying pathological processes in the central nervous system and as an informative adjunct to LM and TEM.

Perspectives on in situ electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zheng, Haimei; Zhu, Yimei

In situ transmission electron microscopy (TEM) with the ability to reveal materials dynamic processes with high spatial and temporal resolution has attracted significant interest. The recent advances in in situ methods, including liquid and gas sample environment, pump-probe ultrafast microscopy, nanomechanics and ferroelectric domain switching the aberration corrected electron optics as well as fast electron detector has opened new opportunities to extend the impact of in situ TEM in broad areas of research ranging from materials science to chemistry, physics and biology. Here in this paper, we highlight the development of liquid environment electron microscopy and its applications in themore » study of colloidal nanoparticle growth, electrochemical processes and others; in situ study of topological vortices in ferroelectric and ferromagnetic materials. At the end, perspectives of future in situ TEM are provided.« less

Perspectives on in situ electron microscopy

DOE PAGES

Zheng, Haimei; Zhu, Yimei

2017-03-29

In situ transmission electron microscopy (TEM) with the ability to reveal materials dynamic processes with high spatial and temporal resolution has attracted significant interest. The recent advances in in situ methods, including liquid and gas sample environment, pump-probe ultrafast microscopy, nanomechanics and ferroelectric domain switching the aberration corrected electron optics as well as fast electron detector has opened new opportunities to extend the impact of in situ TEM in broad areas of research ranging from materials science to chemistry, physics and biology. Here in this paper, we highlight the development of liquid environment electron microscopy and its applications in themore » study of colloidal nanoparticle growth, electrochemical processes and others; in situ study of topological vortices in ferroelectric and ferromagnetic materials. At the end, perspectives of future in situ TEM are provided.« less

A direct electron detector for time-resolved MeV electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vecchione, T.; Denes, P.; Jobe, R. K.

The introduction of direct electron detectors enabled the structural biology revolution of cryogenic electron microscopy. Direct electron detectors are now expected to have a similarly dramatic impact on time-resolved MeV electron microscopy, particularly by enabling both spatial and temporal jitter correction. Here we report on the commissioning of a direct electron detector for time-resolved MeV electron microscopy. The direct electron detector demonstrated MeV single electron sensitivity and is capable of recording megapixel images at 180 Hz. The detector has a 15-bit dynamic range, better than 30-μmμm spatial resolution and less than 20 analogue-to-digital converter count RMS pixel noise. The uniquemore » capabilities of the direct electron detector and the data analysis required to take advantage of these capabilities are presented. The technical challenges associated with generating and processing large amounts of data are also discussed.« less

Electron Microscopy of Ebola Virus-Infected Cells.

PubMed

Noda, Takeshi

2017-01-01

Ebola virus (EBOV) replicates in host cells, where both viral and cellular components show morphological changes during the process of viral replication from entry to budding. These steps in the replication cycle can be studied using electron microscopy (EM), including transmission electron microscopy (TEM) and scanning electron microscopy (SEM), which is one of the most useful methods for visualizing EBOV particles and EBOV-infected cells at the ultrastructural level. This chapter describes conventional methods for EM sample preparation of cultured cells infected with EBOV.

Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jesse, S.; Chi, M.; Belianinov, A.

Electron microscopy is undergoing a transition; from the model of producing only a few micrographs, through the current state where many images and spectra can be digitally recorded, to a new mode where very large volumes of data (movies, ptychographic and multi-dimensional series) can be rapidly obtained. In this paper, we discuss the application of so-called “big-data” methods to high dimensional microscopy data, using unsupervised multivariate statistical techniques, in order to explore salient image features in a specific example of BiFeO 3 domains. Remarkably, k-means clustering reveals domain differentiation despite the fact that the algorithm is purely statistical in naturemore » and does not require any prior information regarding the material, any coexisting phases, or any differentiating structures. While this is a somewhat trivial case, this example signifies the extraction of useful physical and structural information without any prior bias regarding the sample or the instrumental modality. Further interpretation of these types of results may still require human intervention. Finally, however, the open nature of this algorithm and its wide availability, enable broad collaborations and exploratory work necessary to enable efficient data analysis in electron microscopy.« less

Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

DOE PAGES

Jesse, S.; Chi, M.; Belianinov, A.; ...

2016-05-23

Electron microscopy is undergoing a transition; from the model of producing only a few micrographs, through the current state where many images and spectra can be digitally recorded, to a new mode where very large volumes of data (movies, ptychographic and multi-dimensional series) can be rapidly obtained. In this paper, we discuss the application of so-called “big-data” methods to high dimensional microscopy data, using unsupervised multivariate statistical techniques, in order to explore salient image features in a specific example of BiFeO 3 domains. Remarkably, k-means clustering reveals domain differentiation despite the fact that the algorithm is purely statistical in naturemore » and does not require any prior information regarding the material, any coexisting phases, or any differentiating structures. While this is a somewhat trivial case, this example signifies the extraction of useful physical and structural information without any prior bias regarding the sample or the instrumental modality. Further interpretation of these types of results may still require human intervention. Finally, however, the open nature of this algorithm and its wide availability, enable broad collaborations and exploratory work necessary to enable efficient data analysis in electron microscopy.« less

A direct electron detector for time-resolved MeV electron microscopy

DOE PAGES

Vecchione, T.; Denes, P.; Jobe, R. K.; ...

2017-03-15

The introduction of direct electron detectors enabled the structural biology revolution of cryogenic electron microscopy. Direct electron detectors are now expected to have a similarly dramatic impact on time-resolved MeV electron microscopy, particularly by enabling both spatial and temporal jitter correction. Here in this paper, we report on the commissioning of a direct electron detector for time-resolved MeV electron microscopy. The direct electron detector demonstrated MeV single electron sensitivity and is capable of recording megapixel images at 180 Hz. The detector has a 15-bit dynamic range, better than 30-μm spatial resolution and less than 20 analogue-to-digital converter count RMS pixelmore » noise. The unique capabilities of the direct electron detector and the data analysis required to take advantage of these capabilities are presented. The technical challenges associated with generating and processing large amounts of data are also discussed.« less

A direct electron detector for time-resolved MeV electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vecchione, T.; Denes, P.; Jobe, R. K.

The introduction of direct electron detectors enabled the structural biology revolution of cryogenic electron microscopy. Direct electron detectors are now expected to have a similarly dramatic impact on time-resolved MeV electron microscopy, particularly by enabling both spatial and temporal jitter correction. Here in this paper, we report on the commissioning of a direct electron detector for time-resolved MeV electron microscopy. The direct electron detector demonstrated MeV single electron sensitivity and is capable of recording megapixel images at 180 Hz. The detector has a 15-bit dynamic range, better than 30-μm spatial resolution and less than 20 analogue-to-digital converter count RMS pixelmore » noise. The unique capabilities of the direct electron detector and the data analysis required to take advantage of these capabilities are presented. The technical challenges associated with generating and processing large amounts of data are also discussed.« less

Fully Hydrated Yeast Cells Imaged with Electron Microscopy

PubMed Central

Peckys, Diana B.; Mazur, Peter; Gould, Kathleen L.; de Jonge, Niels

2011-01-01

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccaromyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. PMID:21575587

Fully hydrated yeast cells imaged with electron microscopy.

PubMed

Peckys, Diana B; Mazur, Peter; Gould, Kathleen L; de Jonge, Niels

2011-05-18

We demonstrate electron microscopy of fully hydrated eukaryotic cells with nanometer resolution. Living Schizosaccharomyces pombe cells were loaded in a microfluidic chamber and imaged in liquid with scanning transmission electron microscopy (STEM). The native intracellular (ultra)structures of wild-type cells and three different mutants were studied without prior labeling, fixation, or staining. The STEM images revealed various intracellular components that were identified on the basis of their shape, size, location, and mass density. The maximal achieved spatial resolution in this initial study was 32 ± 8 nm, an order of magnitude better than achievable with light microscopy on pristine cells. Light-microscopy images of the same samples were correlated with the corresponding electron-microscopy images. Achieving synergy between the capabilities of light and electron microscopy, we anticipate that liquid STEM will be broadly applied to explore the ultrastructure of live cells. Copyright © 2011 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Electronic Blending in Virtual Microscopy

ERIC Educational Resources Information Center

Maybury, Terrence S.; Farah, Camile S.

2010-01-01

Virtual microscopy (VM) is a relatively new technology that transforms the computer into a microscope. In essence, VM allows for the scanning and transfer of glass slides from light microscopy technology to the digital environment of the computer. This transition is also a function of the change from print knowledge to electronic knowledge, or as…

The importance of transmission electron microscopy analysis of spermatozoa: Diagnostic applications and basic research.

PubMed

Moretti, Elena; Sutera, Gaetano; Collodel, Giulia

2016-06-01

This review is aimed at discussing the role of ultrastructural studies on human spermatozoa and evaluating transmission electron microscopy as a diagnostic tool that can complete andrology protocols. It is clear that morphological sperm defects may explain decreased fertilizing potential and acquire particular value in the field of male infertility. Electron microscopy is the best method to identify systematic or monomorphic and non-systematic or polymorphic sperm defects. The systematic defects are characterized by a particular anomaly that affects the vast majority of spermatozoa in a semen sample, whereas a heterogeneous combination of head and tail defects found in variable percentages are typically non-systematic or polymorphic sperm defects. A correct diagnosis of these specific sperm alterations is important for choosing the male infertility's therapy and for deciding to turn to assisted reproduction techniques. Transmission electron microscopy (TEM) also represents a valuable method to explore the in vitro effects of different compounds (for example drugs with potential spermicidal activity) on the morphology of human spermatozoa. Finally, TEM used in combination with immunohistochemical techniques, integrates structural and functional aspects that provide a wide horizon in the understanding of sperm physiology and pathology. transmission electron microscopy: TEM; World Health Organization: WHO; light microscopy: LM; motile sperm organelle morphology examination: MSOME; intracytoplasmic morphologically selected sperm injection: IMSI; intracytoplasmic sperm injection: ICSI; dysplasia of fibrous sheath: DFS; primary ciliary dyskinesia: PCD; outer dense fibers: ODF; assisted reproduction technologies: ART; scanning electron microscopy: SEM; polyvinylpirrolidone: PVP; tert-butylhydroperoxide: TBHP.

Sparse imaging for fast electron microscopy

NASA Astrophysics Data System (ADS)

Anderson, Hyrum S.; Ilic-Helms, Jovana; Rohrer, Brandon; Wheeler, Jason; Larson, Kurt

2013-02-01

Scanning electron microscopes (SEMs) are used in neuroscience and materials science to image centimeters of sample area at nanometer scales. Since imaging rates are in large part SNR-limited, large collections can lead to weeks of around-the-clock imaging time. To increase data collection speed, we propose and demonstrate on an operational SEM a fast method to sparsely sample and reconstruct smooth images. To accurately localize the electron probe position at fast scan rates, we model the dynamics of the scan coils, and use the model to rapidly and accurately visit a randomly selected subset of pixel locations. Images are reconstructed from the undersampled data by compressed sensing inversion using image smoothness as a prior. We report image fidelity as a function of acquisition speed by comparing traditional raster to sparse imaging modes. Our approach is equally applicable to other domains of nanometer microscopy in which the time to position a probe is a limiting factor (e.g., atomic force microscopy), or in which excessive electron doses might otherwise alter the sample being observed (e.g., scanning transmission electron microscopy).

Dynamic imaging with electron microscopy

ScienceCinema

Campbell, Geoffrey; McKeown, Joe; Santala, Melissa

2018-02-13

Livermore researchers have perfected an electron microscope to study fast-evolving material processes and chemical reactions. By applying engineering, microscopy, and laser expertise to the decades-old technology of electron microscopy, the dynamic transmission electron microscope (DTEM) team has developed a technique that can capture images of phenomena that are both very small and very fast. DTEM uses a precisely timed laser pulse to achieve a short but intense electron beam for imaging. When synchronized with a dynamic event in the microscope's field of view, DTEM allows scientists to record and measure material changes in action. A new movie-mode capability, which earned a 2013 R&D 100 Award from R&D Magazine, uses up to nine laser pulses to sequentially capture fast, irreversible, even one-of-a-kind material changes at the nanometer scale. DTEM projects are advancing basic and applied materials research, including such areas as nanostructure growth, phase transformations, and chemical reactions.

Nano-fEM: protein localization using photo-activated localization microscopy and electron microscopy.

PubMed

Watanabe, Shigeki; Richards, Jackson; Hollopeter, Gunther; Hobson, Robert J; Davis, Wayne M; Jorgensen, Erik M

2012-12-03

Mapping the distribution of proteins is essential for understanding the function of proteins in a cell. Fluorescence microscopy is extensively used for protein localization, but subcellular context is often absent in fluorescence images. Immuno-electron microscopy, on the other hand, can localize proteins, but the technique is limited by a lack of compatible antibodies, poor preservation of morphology and because most antigens are not exposed to the specimen surface. Correlative approaches can acquire the fluorescence image from a whole cell first, either from immuno-fluorescence or genetically tagged proteins. The sample is then fixed and embedded for electron microscopy, and the images are correlated (1-3). However, the low-resolution fluorescence image and the lack of fiducial markers preclude the precise localization of proteins. Alternatively, fluorescence imaging can be done after preserving the specimen in plastic. In this approach, the block is sectioned, and fluorescence images and electron micrographs of the same section are correlated (4-7). However, the diffraction limit of light in the correlated image obscures the locations of individual molecules, and the fluorescence often extends beyond the boundary of the cell. Nano-resolution fluorescence electron microscopy (nano-fEM) is designed to localize proteins at nano-scale by imaging the same sections using photo-activated localization microscopy (PALM) and electron microscopy. PALM overcomes the diffraction limit by imaging individual fluorescent proteins and subsequently mapping the centroid of each fluorescent spot (8-10). We outline the nano-fEM technique in five steps. First, the sample is fixed and embedded using conditions that preserve the fluorescence of tagged proteins. Second, the resin blocks are sectioned into ultrathin segments (70-80 nm) that are mounted on a cover glass. Third, fluorescence is imaged in these sections using the Zeiss PALM microscope. Fourth, electron dense structures are

Hybrid fluorescence and electron cryo-microscopy for simultaneous electron and photon imaging.

PubMed

Iijima, Hirofumi; Fukuda, Yoshiyuki; Arai, Yoshihiro; Terakawa, Susumu; Yamamoto, Naoki; Nagayama, Kuniaki

2014-01-01

Integration of fluorescence light and transmission electron microscopy into the same device would represent an important advance in correlative microscopy, which traditionally involves two separate microscopes for imaging. To achieve such integration, the primary technical challenge that must be solved regards how to arrange two objective lenses used for light and electron microscopy in such a manner that they can properly focus on a single specimen. To address this issue, both lateral displacement of the specimen between two lenses and specimen rotation have been proposed. Such movement of the specimen allows sequential collection of two kinds of microscopic images of a single target, but prevents simultaneous imaging. This shortcoming has been made up by using a simple optical device, a reflection mirror. Here, we present an approach toward the versatile integration of fluorescence and electron microscopy for simultaneous imaging. The potential of simultaneous hybrid microscopy was demonstrated by fluorescence and electron sequential imaging of a fluorescent protein expressed in cells and cathodoluminescence imaging of fluorescent beads. Copyright © 2013 Elsevier Inc. All rights reserved.

Correlative Fluorescence and Electron Microscopy

PubMed Central

Schirra, Randall T.; Zhang, Peijun

2014-01-01

Correlative fluorescence and electron microscopy (CFEM) is a multimodal technique that combines dynamic and localization information from fluorescence methods with ultrastructural data from electron microscopy, to give new information about how cellular components change relative to the spatiotemporal dynamics within their environment. In this review, we will discuss some of the basic techniques and tools of the trade for utilizing this attractive research method, which is becoming a very powerful tool for biology labs. The information obtained from correlative methods has proven to be invaluable in creating consensus between the two types of microscopy, extending the capability of each, and cutting the time and expense associate with using each method separately for comparative analysis. The realization of the advantages of these methods in cell biology have led to rapid improvement in the protocols and have ushered in a new generation of instruments to reach the next level of correlation – integration. PMID:25271959

Attomicroscopy: from femtosecond to attosecond electron microscopy

NASA Astrophysics Data System (ADS)

Hassan, Mohammed Th

2018-02-01

In the last decade, the development of ultrafast electron diffraction (UED) and microscopy (UEM) have enabled the imaging of atomic motion in real time and space. These pivotal table-top tools opened the door for a vast range of applications in different areas of science spanning chemistry, physics, materials science, and biology. We first discuss the basic principles and recent advancements, including some of the important applications, of both UED and UEM. Then, we discuss the recent advances in the field that have enhanced the spatial and temporal resolutions, where the latter, is however, still limited to a few hundreds of femtoseconds, preventing the imaging of ultrafast dynamics of matter lasting few tens of femtoseconds. Then, we present our new optical gating approach for generating an isolated 30 fs electron pulse with sufficient intensity to attain a temporal resolution on the same time scale. This achievement allows, for the first time, imaging the electron dynamics of matter. Finally, we demonstrate the feasibility of the optical gating approach to generate an isolated attosecond electron pulse, utilizing our recently demonstrated optical attosecond laser pulse, which paves the way for establishing the field of ‘Attomicroscopy’, ultimately enabling us to image the electron motion in action.

The origins and evolution of freeze-etch electron microscopy

PubMed Central

Heuser, John E.

2011-01-01

The introduction of the Balzers freeze-fracture machine by Moor in 1961 had a much greater impact on the advancement of electron microscopy than he could have imagined. Devised originally to circumvent the dangers of classical thin-section techniques, as well as to provide unique en face views of cell membranes, freeze-fracturing proved to be crucial for developing modern concepts of how biological membranes are organized and proved that membranes are bilayers of lipids within which proteins float and self-assemble. Later, when freeze-fracturing was combined with methods for freezing cells that avoided the fixation and cryoprotection steps that Moor still had to use to prepare the samples for his original invention, it became a means for capturing membrane dynamics on the millisecond time-scale, thus allowing a deeper understanding of the functions of biological membranes in living cells as well as their static ultrastructure. Finally, the realization that unfixed, non-cryoprotected samples could be deeply vacuum-etched or even freeze-dried after freeze-fracturing opened up a whole new way to image all the other molecular components of cells besides their membranes and also provided a powerful means to image the interactions of all the cytoplasmic components with the various membranes of the cell. The purpose of this review is to outline the history of these technical developments, to describe how they are being used in electron microscopy today and to suggest how they can be improved in order to further their utility for biological electron microscopy in the future. PMID:21844598

A correlative optical microscopy and scanning electron microscopy approach to locating nanoparticles in brain tumors.

PubMed

Kempen, Paul J; Kircher, Moritz F; de la Zerda, Adam; Zavaleta, Cristina L; Jokerst, Jesse V; Mellinghoff, Ingo K; Gambhir, Sanjiv S; Sinclair, Robert

2015-01-01

The growing use of nanoparticles in biomedical applications, including cancer diagnosis and treatment, demands the capability to exactly locate them within complex biological systems. In this work a correlative optical and scanning electron microscopy technique was developed to locate and observe multi-modal gold core nanoparticle accumulation in brain tumor models. Entire brain sections from mice containing orthotopic brain tumors injected intravenously with nanoparticles were imaged using both optical microscopy to identify the brain tumor, and scanning electron microscopy to identify the individual nanoparticles. Gold-based nanoparticles were readily identified in the scanning electron microscope using backscattered electron imaging as bright spots against a darker background. This information was then correlated to determine the exact location of the nanoparticles within the brain tissue. The nanoparticles were located only in areas that contained tumor cells, and not in the surrounding healthy brain tissue. This correlative technique provides a powerful method to relate the macro- and micro-scale features visible in light microscopy with the nanoscale features resolvable in scanning electron microscopy. Copyright © 2014 Elsevier Ltd. All rights reserved.

High-resolution imaging by scanning electron microscopy of semithin sections in correlation with light microscopy.

PubMed

Koga, Daisuke; Kusumi, Satoshi; Shodo, Ryusuke; Dan, Yukari; Ushiki, Tatsuo

2015-12-01

In this study, we introduce scanning electron microscopy (SEM) of semithin resin sections. In this technique, semithin sections were adhered on glass slides, stained with both uranyl acetate and lead citrate, and observed with a backscattered electron detector at a low accelerating voltage. As the specimens are stained in the same manner as conventional transmission electron microscopy (TEM), the contrast of SEM images of semithin sections was similar to TEM images of ultrathin sections. Using this technique, wide areas of semithin sections were also observed by SEM, without the obstruction of grids, which was inevitable for traditional TEM. This study also applied semithin section SEM to correlative light and electron microscopy. Correlative immunofluorescence microscopy and immune-SEM were performed in semithin sections of LR white resin-embedded specimens using a FluoroNanogold-labeled secondary antibody. Because LR white resin is hydrophilic and electron stable, this resin is suitable for immunostaining and SEM observation. Using correlative microscopy, the precise localization of the primary antibody was demonstrated by fluorescence microscopy and SEM. This method has great potential for studies examining the precise localization of molecules, including Golgi- and ER-associated proteins, in correlation with LM and SEM. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Platinum replica electron microscopy: Imaging the cytoskeleton globally and locally.

PubMed

Svitkina, Tatyana M

2017-05-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the "comfort zones" of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. Copyright © 2017 Elsevier Ltd. All rights reserved.

Platinum Replica Electron Microscopy: Imaging the Cytoskeleton Globally and Locally

PubMed Central

SVITKINA, Tatyana M.

2017-01-01

Structural studies reveal how smaller components of a system work together as a whole. However, combining high resolution of details with full coverage of the whole is challenging. In cell biology, light microscopy can image many cells in their entirety, but at a lower resolution, whereas electron microscopy affords very high resolution, but usually at the expense of the sample size and coverage. Structural analyses of the cytoskeleton are especially demanding, because cytoskeletal networks are unresolvable by light microscopy due to their density and intricacy, whereas their proper preservation is a challenge for electron microscopy. Platinum replica electron microscopy can uniquely bridge the gap between the “comfort zones” of light and electron microscopy by allowing high resolution imaging of the cytoskeleton throughout the entire cell and in many cells in the population. This review describes the principles and applications of platinum replica electron microscopy for studies of the cytoskeleton. PMID:28323208

Environmental scanning electron microscopy in cell biology.

PubMed

McGregor, J E; Staniewicz, L T L; Guthrie Neé Kirk, S E; Donald, A M

2013-01-01

Environmental scanning electron microscopy (ESEM) (1) is an imaging technique which allows hydrated, insulating samples to be imaged under an electron beam. The resolution afforded by this technique is higher than conventional optical microscopy but lower than conventional scanning electron microscopy (CSEM). The major advantage of the technique is the minimal sample preparation needed, making ESEM quick to use and the images less susceptible to the artifacts that the extensive sample preparation usually required for CSEM may introduce. Careful manipulation of both the humidity in the microscope chamber and the beam energy are nevertheless essential to prevent dehydration and beam damage artifacts. In some circumstances it is possible to image live cells in the ESEM (2).In the following sections we introduce the fundamental principles of ESEM imaging before presenting imaging protocols for plant epidermis, mammalian cells, and bacteria. In the first two cases samples are imaged using the secondary electron (topographic) signal, whereas a transmission technique is employed to image bacteria.

Electron microscopy investigations of nanoparticles for cancer diagnostic applications

NASA Astrophysics Data System (ADS)

Koh, Ai Leen

This dissertation concerns electron microscopy characterization of magnetic (MNP) and surface enhanced Raman scattering (SERS) nanoparticles for in-vitro cancer diagnostic applications. Electron microscopy is an essential characterization tool owing to its (sub) nanometer spatial resolution. Structural information about the nanoparticles can be obtained using transmission electron microscopy (TEM), which can in turn be correlated to their physical characteristics. The scanning electron microscope (SEM) has excellent depth of field and can be effectively utilized to obtain high resolution information about nanoparticles binding onto cell surfaces. Part One of this thesis focuses on MNPs for bio-sensing and detection applications. As a preliminary study, chemically-synthesized, commercially-available iron oxide nanoparticles were compared against their laboratory-synthesized counterparts to assess their suitability for this application. The motivation for this initial study came about due to the lack of published data on commercially available iron oxide nanoparticles. TEM studies show that the latter are "beads" composed of multiple iron oxide cores encapsulated by a polymer shell, with large standard deviations in core diameter. Laboratory-synthesized iron oxide nanoparticles, on the other hand, are single core particles with small variations in diameter and therefore are expected to be better candidates for the required application. A key limitation in iron oxide nanoparticles is their relatively weak magnetic signals. The development of high moment Synthetic Anti-Ferromagnetic (SAF) nanoparticles aims to overcome this issue. SAFs are a novel class of MNPs fabricated using nanoimprint lithography, direct deposition of multilayer structure and final suspension into liquid medium (water). TEM analyses of cross-section specimens reveal that the SAFs possess characteristics similar to those of sputtered magnetic multilayer thin films. Their layered structure is

Quantifying the cellular uptake of semiconductor quantum dot nanoparticles by analytical electron microscopy.

PubMed

Hondow, Nicole; Brown, M Rowan; Starborg, Tobias; Monteith, Alexander G; Brydson, Rik; Summers, Huw D; Rees, Paul; Brown, Andy

2016-02-01

Semiconductor quantum dot nanoparticles are in demand as optical biomarkers yet the cellular uptake process is not fully understood; quantification of numbers and the fate of internalized particles are still to be achieved. We have focussed on the characterization of cellular uptake of quantum dots using a combination of analytical electron microscopies because of the spatial resolution available to examine uptake at the nanoparticle level, using both imaging to locate particles and spectroscopy to confirm identity. In this study, commercially available quantum dots, CdSe/ZnS core/shell particles coated in peptides to target cellular uptake by endocytosis, have been investigated in terms of the agglomeration state in typical cell culture media, the traverse of particle agglomerates across U-2 OS cell membranes during endocytosis, the merging of endosomal vesicles during incubation of cells and in the correlation of imaging flow cytometry and transmission electron microscopy to measure the final nanoparticle dose internalized by the U-2 OS cells. We show that a combination of analytical transmission electron microscopy and serial block face scanning electron microscopy can provide a comprehensive description of the internalization of an initial exposure dose of nanoparticles by an endocytically active cell population and how the internalized, membrane bound nanoparticle load is processed by the cells. We present a stochastic model of an endosome merging process and show that this provides a data-driven modelling framework for the prediction of cellular uptake of engineered nanoparticles in general. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

The significance of Bragg's law in electron diffraction and microscopy, and Bragg's second law.

PubMed

Humphreys, C J

2013-01-01

Bragg's second law, which deserves to be more widely known, is recounted. The significance of Bragg's law in electron diffraction and microscopy is then discussed, with particular emphasis on differences between X-ray and electron diffraction. As an example of such differences, the critical voltage effect in electron diffraction is described. It is then shown that the lattice imaging of crystals in high-resolution electron microscopy directly reveals the Bragg planes used for the imaging process, exactly as visualized by Bragg in his real-space law. Finally, it is shown how in 2012, for the first time, on the centennial anniversary of Bragg's law, single atoms have been identified in an electron microscope using X-rays emitted from the specimen. Hence atomic resolution X-ray maps of a crystal in real space can be formed which give the positions and identities of the different atoms in the crystal, or of a single impurity atom in the crystal.

Correlative fluorescence microscopy and scanning transmission electron microscopy of quantum-dot-labeled proteins in whole cells in liquid.

PubMed

Dukes, Madeline J; Peckys, Diana B; de Jonge, Niels

2010-07-27

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7x12 nm were visible in a 5 microm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs.

Diffraction and microscopy with attosecond electron pulse trains

NASA Astrophysics Data System (ADS)

Morimoto, Yuya; Baum, Peter

2018-03-01

Attosecond spectroscopy1-7 can resolve electronic processes directly in time, but a movie-like space-time recording is impeded by the too long wavelength ( 100 times larger than atomic distances) or the source-sample entanglement in re-collision techniques8-11. Here we advance attosecond metrology to picometre wavelength and sub-atomic resolution by using free-space electrons instead of higher-harmonic photons1-7 or re-colliding wavepackets8-11. A beam of 70-keV electrons at 4.5-pm de Broglie wavelength is modulated by the electric field of laser cycles into a sequence of electron pulses with sub-optical-cycle duration. Time-resolved diffraction from crystalline silicon reveals a < 10-as delay of Bragg emission and demonstrates the possibility of analytic attosecond-ångström diffraction. Real-space electron microscopy visualizes with sub-light-cycle resolution how an optical wave propagates in space and time. This unification of attosecond science with electron microscopy and diffraction enables space-time imaging of light-driven processes in the entire range of sample morphologies that electron microscopy can access.

Fast electron microscopy via compressive sensing

DOEpatents

Larson, Kurt W; Anderson, Hyrum S; Wheeler, Jason W

2014-12-09

Various technologies described herein pertain to compressive sensing electron microscopy. A compressive sensing electron microscope includes a multi-beam generator and a detector. The multi-beam generator emits a sequence of electron patterns over time. Each of the electron patterns can include a plurality of electron beams, where the plurality of electron beams is configured to impart a spatially varying electron density on a sample. Further, the spatially varying electron density varies between each of the electron patterns in the sequence. Moreover, the detector collects signals respectively corresponding to interactions between the sample and each of the electron patterns in the sequence.

Quantitative Scanning Transmission Electron Microscopy of Electronic and Nanostructured Materials

NASA Astrophysics Data System (ADS)

Yankovich, Andrew B.

Electronic and nanostructured materials have been investigated using advanced scanning transmission electron microscopy (STEM) techniques. The first topic is the microstructure of Ga and Sb-doped ZnO. Ga-doped ZnO is a candidate transparent conducting oxide material. The microstructure of GZO thin films grown by MBE under different growth conditions and different substrates were examined using various electron microscopy (EM) techniques. The microstructure, prevalent defects, and polarity in these films strongly depend on the growth conditions and substrate. Sb-doped ZnO nanowires have been shown to be the first route to stable p-type ZnO. Using Z-contrast STEM, I have showed that an unusual microstructure of Sb-decorated head-to-head inversion domain boundaries and internal voids contain all the Sb in the nanowires and cause the p-type conduction. InGaN thin films and InGaN / GaN quantum wells (QW) for light emitting diodes are the second topic. Low-dose Z-contrast STEM, PACBED, and EDS on InGaN QW LED structures grown by MOCVD show no evidence for nanoscale composition variations, contradicting previous reports. In addition, a new extended defect in GaN and InGaN was discovered. The defect consists of a faceted pyramid-shaped void that produces a threading dislocation along the [0001] growth direction, and is likely caused by carbon contamination during growth. Non-rigid registration (NRR) and high-precision STEM of nanoparticles is the final topic. NRR is a new image processing technique that corrects distortions arising from the serial nature of STEM acquisition that previously limited the precision of locating atomic columns and counting the number of atoms in images. NRR was used to demonstrate sub-picometer precision in STEM images of single crystal Si and GaN, the best achieved in EM. NRR was used to measure the atomic surface structure of Pt nanoacatalysts and Au nanoparticles, which revealed new bond length variation phenomenon of surface atoms. In

Correlative Fluorescence Microscopy and Scanning Transmission Electron Microscopy of Quantum Dot Labeled Proteins in Whole Cells in Liquid

PubMed Central

Dukes, Madeline J.; Peckys, Diana B.; de Jonge, Niels

2010-01-01

Correlative fluorescence microscopy and transmission electron microscopy (TEM) is a state-of-the-art microscopy methodology to study cellular function, combining the functionality of light microscopy with the high resolution of electron microscopy. However, this technique involves complex sample preparation procedures due to its need for either thin sections or frozen samples for TEM imaging. Here, we introduce a novel correlative approach capable of imaging whole eukaryotic cells in liquid with fluorescence microscopy and with scanning transmission electron microscopy (STEM); there is no additional sample preparation necessary for the electron microscopy. Quantum dots (QDs) were bound to epidermal growth factor (EGF) receptors of COS7 fibroblast cells. Fixed whole cells in saline water were imaged with fluorescence microscopy and subsequently with STEM. The STEM images were correlated with fluorescence images of the same cellular regions. QDs of dimensions 7 × 12 nm were visible in a 5 μm thick layer of saline water, consistent with calculations. A spatial resolution of 3 nm was achieved on the QDs. PMID:20550177

New developments in electron microscopy for serial image acquisition of neuronal profiles.

PubMed

Kubota, Yoshiyuki

2015-02-01

Recent developments in electron microscopy largely automate the continuous acquisition of serial electron micrographs (EMGs), previously achieved by laborious manual serial ultrathin sectioning using an ultramicrotome and ultrastructural image capture process with transmission electron microscopy. The new systems cut thin sections and capture serial EMGs automatically, allowing for acquisition of large data sets in a reasonably short time. The new methods are focused ion beam/scanning electron microscopy, ultramicrotome/serial block-face scanning electron microscopy, automated tape-collection ultramicrotome/scanning electron microscopy and transmission electron microscope camera array. In this review, their positive and negative aspects are discussed. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Photon gating in four-dimensional ultrafast electron microscopy.

PubMed

Hassan, Mohammed T; Liu, Haihua; Baskin, John Spencer; Zewail, Ahmed H

2015-10-20

Ultrafast electron microscopy (UEM) is a pivotal tool for imaging of nanoscale structural dynamics with subparticle resolution on the time scale of atomic motion. Photon-induced near-field electron microscopy (PINEM), a key UEM technique, involves the detection of electrons that have gained energy from a femtosecond optical pulse via photon-electron coupling on nanostructures. PINEM has been applied in various fields of study, from materials science to biological imaging, exploiting the unique spatial, energy, and temporal characteristics of the PINEM electrons gained by interaction with a "single" light pulse. The further potential of photon-gated PINEM electrons in probing ultrafast dynamics of matter and the optical gating of electrons by invoking a "second" optical pulse has previously been proposed and examined theoretically in our group. Here, we experimentally demonstrate this photon-gating technique, and, through diffraction, visualize the phase transition dynamics in vanadium dioxide nanoparticles. With optical gating of PINEM electrons, imaging temporal resolution was improved by a factor of 3 or better, being limited only by the optical pulse widths. This work enables the combination of the high spatial resolution of electron microscopy and the ultrafast temporal response of the optical pulses, which provides a promising approach to attain the resolution of few femtoseconds and attoseconds in UEM.

Photon gating in four-dimensional ultrafast electron microscopy

PubMed Central

Hassan, Mohammed T.; Liu, Haihua; Baskin, John Spencer; Zewail, Ahmed H.

2015-01-01

Ultrafast electron microscopy (UEM) is a pivotal tool for imaging of nanoscale structural dynamics with subparticle resolution on the time scale of atomic motion. Photon-induced near-field electron microscopy (PINEM), a key UEM technique, involves the detection of electrons that have gained energy from a femtosecond optical pulse via photon–electron coupling on nanostructures. PINEM has been applied in various fields of study, from materials science to biological imaging, exploiting the unique spatial, energy, and temporal characteristics of the PINEM electrons gained by interaction with a “single” light pulse. The further potential of photon-gated PINEM electrons in probing ultrafast dynamics of matter and the optical gating of electrons by invoking a “second” optical pulse has previously been proposed and examined theoretically in our group. Here, we experimentally demonstrate this photon-gating technique, and, through diffraction, visualize the phase transition dynamics in vanadium dioxide nanoparticles. With optical gating of PINEM electrons, imaging temporal resolution was improved by a factor of 3 or better, being limited only by the optical pulse widths. This work enables the combination of the high spatial resolution of electron microscopy and the ultrafast temporal response of the optical pulses, which provides a promising approach to attain the resolution of few femtoseconds and attoseconds in UEM. PMID:26438835

Copper Decoration of Carbon Nanotubes and High Resolution Electron Microscopy

NASA Astrophysics Data System (ADS)

Probst, Camille

A new process of decorating carbon nanotubes with copper was developed for the fabrication of nanocomposite aluminum-nanotubes. The process consists of three stages: oxidation, activation and electroless copper plating on the nanotubes. The oxidation step was required to create chemical function on the nanotubes, essential for the activation step. Then, catalytic nanoparticles of tin-palladium were deposited on the tubes. Finally, during the electroless copper plating, copper particles with a size between 20 and 60 nm were uniformly deposited on the nanotubes surface. The reproducibility of the process was shown by using another type of carbon nanotube. The fabrication of nanocomposites aluminum-nanotubes was tested by aluminum vacuum infiltration. Although the infiltration of carbon nanotubes did not produce the expected results, an interesting electron microscopy sample was discovered during the process development: the activated carbon nanotubes. Secondly, scanning transmitted electron microscopy (STEM) imaging in SEM was analysed. The images were obtained with a new detector on the field emission scanning electron microscope (Hitachi S-4700). Various parameters were analysed with the use of two different samples: the activated carbon nanotubes (previously obtained) and gold-palladium nanodeposits. Influences of working distance, accelerating voltage or sample used on the spatial resolution of images obtained with SMART (Scanning Microscope Assessment and Resolution Testing) were analysed. An optimum working distance for the best spatial resolution related to the sample analysed was found for the imaging in STEM mode. Finally, relation between probe size and spatial resolution of backscattered electrons (BSE) images was studied. An image synthesis method was developed to generate the BSE images from backscattered electrons coefficients obtained with CASINO software. Spatial resolution of images was determined using SMART. The analysis shown that using a probe

Simultaneous Correlative Scanning Electron and High-NA Fluorescence Microscopy

PubMed Central

Liv, Nalan; Zonnevylle, A. Christiaan; Narvaez, Angela C.; Effting, Andries P. J.; Voorneveld, Philip W.; Lucas, Miriam S.; Hardwick, James C.; Wepf, Roger A.; Kruit, Pieter; Hoogenboom, Jacob P.

2013-01-01

Correlative light and electron microscopy (CLEM) is a unique method for investigating biological structure-function relations. With CLEM protein distributions visualized in fluorescence can be mapped onto the cellular ultrastructure measured with electron microscopy. Widespread application of correlative microscopy is hampered by elaborate experimental procedures related foremost to retrieving regions of interest in both modalities and/or compromises in integrated approaches. We present a novel approach to correlative microscopy, in which a high numerical aperture epi-fluorescence microscope and a scanning electron microscope illuminate the same area of a sample at the same time. This removes the need for retrieval of regions of interest leading to a drastic reduction of inspection times and the possibility for quantitative investigations of large areas and datasets with correlative microscopy. We demonstrate Simultaneous CLEM (SCLEM) analyzing cell-cell connections and membrane protrusions in whole uncoated colon adenocarcinoma cell line cells stained for actin and cortactin with AlexaFluor488. SCLEM imaging of coverglass-mounted tissue sections with both electron-dense and fluorescence staining is also shown. PMID:23409024

Electron microscopy methods in studies of cultural heritage sites

NASA Astrophysics Data System (ADS)

Vasiliev, A. L.; Kovalchuk, M. V.; Yatsishina, E. B.

2016-11-01

The history of the development and application of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray microanalysis (EDXMA) in studies of cultural heritage sites is considered. In fact, investigations based on these methods began when electron microscopes became a commercial product. Currently, these methods, being developed and improved, help solve many historical enigmas. To date, electron microscopy combined with microanalysis makes it possible to investigate any object, from parchment and wooden articles to pigments, tools, and objects of art. Studies by these methods have revealed that some articles were made by ancient masters using ancient "nanotechnologies"; hence, their comprehensive analysis calls for the latest achievements in the corresponding instrumental methods and sample preparation techniques.

3D correlative light and electron microscopy of cultured cells using serial blockface scanning electron microscopy

PubMed Central

Lerner, Thomas R.; Burden, Jemima J.; Nkwe, David O.; Pelchen-Matthews, Annegret; Domart, Marie-Charlotte; Durgan, Joanne; Weston, Anne; Jones, Martin L.; Peddie, Christopher J.; Carzaniga, Raffaella; Florey, Oliver; Marsh, Mark; Gutierrez, Maximiliano G.

2017-01-01

ABSTRACT The processes of life take place in multiple dimensions, but imaging these processes in even three dimensions is challenging. Here, we describe a workflow for 3D correlative light and electron microscopy (CLEM) of cell monolayers using fluorescence microscopy to identify and follow biological events, combined with serial blockface scanning electron microscopy to analyse the underlying ultrastructure. The workflow encompasses all steps from cell culture to sample processing, imaging strategy, and 3D image processing and analysis. We demonstrate successful application of the workflow to three studies, each aiming to better understand complex and dynamic biological processes, including bacterial and viral infections of cultured cells and formation of entotic cell-in-cell structures commonly observed in tumours. Our workflow revealed new insight into the replicative niche of Mycobacterium tuberculosis in primary human lymphatic endothelial cells, HIV-1 in human monocyte-derived macrophages, and the composition of the entotic vacuole. The broad application of this 3D CLEM technique will make it a useful addition to the correlative imaging toolbox for biomedical research. PMID:27445312

Electron transparent graphene windows for environmental scanning electron microscopy in liquids and dense gases.

PubMed

Stoll, Joshua D; Kolmakov, Andrei

2012-12-21

Due to its ultrahigh electron transmissivity in a wide electron energy range, molecular impermeability, high electrical conductivity and excellent mechanical stiffness, suspended graphene membranes appear to be a nearly ideal window material for in situ (in vivo) environmental electron microscopy of nano- and mesoscopic objects (including bio-medical samples) immersed in liquids and/or in dense gaseous media. In this paper, taking advantage of a small modification of the graphene transfer protocol onto metallic and SiN supporting orifices, reusable environmental cells with exchangeable graphene windows have been designed. Using colloidal gold nanoparticles (50 nm) dispersed in water as model objects for scanning electron microscopy in liquids as proof of concept, different conditions for imaging through the graphene membrane were tested. Limiting factors for electron microscopy in liquids, such as electron beam induced water radiolysis and damage of the graphene membrane at high electron doses, are discussed.

Scanning Transmission Electron Microscopy | Materials Science | NREL

Science.gov Websites

mode by collecting the EDS and EELS signals point-by-point as one scans the electron probe across the . Examples of Scanning Transmission Electron Microscopy Capabilities Z-contrast image microphoto taken by

Phase-contrast scanning transmission electron microscopy.

PubMed

Minoda, Hiroki; Tamai, Takayuki; Iijima, Hirofumi; Hosokawa, Fumio; Kondo, Yukihito

2015-06-01

This report introduces the first results obtained using phase-contrast scanning transmission electron microscopy (P-STEM). A carbon-film phase plate (PP) with a small center hole is placed in the condenser aperture plane so that a phase shift is introduced in the incident electron waves except those passing through the center hole. A cosine-type phase-contrast transfer function emerges when the phase-shifted scattered waves interfere with the non-phase-shifted unscattered waves, which passed through the center hole before incidence onto the specimen. The phase contrast resulting in P-STEM is optically identical to that in phase-contrast transmission electron microscopy that is used to provide high contrast for weak phase objects. Therefore, the use of PPs can enhance the phase contrast of the STEM images of specimens in principle. The phase shift resulting from the PP, whose thickness corresponds to a phase shift of π, has been confirmed using interference fringes displayed in the Ronchigram of a silicon single crystal specimen. The interference fringes were found to abruptly shift at the edge of the PP hole by π. © The Author 2015. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Direct imaging detectors for electron microscopy

NASA Astrophysics Data System (ADS)

Faruqi, A. R.; McMullan, G.

2018-01-01

Electronic detectors used for imaging in electron microscopy are reviewed in this paper. Much of the detector technology is based on the developments in microelectronics, which have allowed the design of direct detectors with fine pixels, fast readout and which are sufficiently radiation hard for practical use. Detectors included in this review are hybrid pixel detectors, monolithic active pixel sensors based on CMOS technology and pnCCDs, which share one important feature: they are all direct imaging detectors, relying on directly converting energy in a semiconductor. Traditional methods of recording images in the electron microscope such as film and CCDs, are mentioned briefly along with a more detailed description of direct electronic detectors. Many applications benefit from the use of direct electron detectors and a few examples are mentioned in the text. In recent years one of the most dramatic advances in structural biology has been in the deployment of the new backthinned CMOS direct detectors to attain near-atomic resolution molecular structures with electron cryo-microscopy (cryo-EM). The development of direct detectors, along with a number of other parallel advances, has seen a very significant amount of new information being recorded in the images, which was not previously possible-and this forms the main emphasis of the review.

Electron microscopy methods in studies of cultural heritage sites

DOE Office of Scientific and Technical Information (OSTI.GOV)

Vasiliev, A. L., E-mail: a.vasiliev56@gmail.com; Kovalchuk, M. V.; Yatsishina, E. B.

The history of the development and application of scanning electron microscopy (SEM), transmission electron microscopy (TEM), and energy-dispersive X-ray microanalysis (EDXMA) in studies of cultural heritage sites is considered. In fact, investigations based on these methods began when electron microscopes became a commercial product. Currently, these methods, being developed and improved, help solve many historical enigmas. To date, electron microscopy combined with microanalysis makes it possible to investigate any object, from parchment and wooden articles to pigments, tools, and objects of art. Studies by these methods have revealed that some articles were made by ancient masters using ancient “nanotechnologies”; hence,more » their comprehensive analysis calls for the latest achievements in the corresponding instrumental methods and sample preparation techniques.« less

Correlative 3D imaging of Whole Mammalian Cells with Light and Electron Microscopy

PubMed Central

Murphy, Gavin E.; Narayan, Kedar; Lowekamp, Bradley C.; Hartnell, Lisa M.; Heymann, Jurgen A. W.; Fu, Jing; Subramaniam, Sriram

2011-01-01

We report methodological advances that extend the current capabilities of ion-abrasion scanning electron microscopy (IA–SEM), also known as focused ion beam scanning electron microscopy, a newly emerging technology for high resolution imaging of large biological specimens in 3D. We establish protocols that enable the routine generation of 3D image stacks of entire plastic-embedded mammalian cells by IA-SEM at resolutions of ~10 to 20 nm at high contrast and with minimal artifacts from the focused ion beam. We build on these advances by describing a detailed approach for carrying out correlative live confocal microscopy and IA–SEM on the same cells. Finally, we demonstrate that by combining correlative imaging with newly developed tools for automated image processing, small 100 nm-sized entities such as HIV-1 or gold beads can be localized in SEM image stacks of whole mammalian cells. We anticipate that these methods will add to the arsenal of tools available for investigating mechanisms underlying host-pathogen interactions, and more generally, the 3D subcellular architecture of mammalian cells and tissues. PMID:21907806

On the Progress of Scanning Transmission Electron Microscopy (STEM) Imaging in a Scanning Electron Microscope.

PubMed

Sun, Cheng; Müller, Erich; Meffert, Matthias; Gerthsen, Dagmar

2018-04-01

Transmission electron microscopy (TEM) with low-energy electrons has been recognized as an important addition to the family of electron microscopies as it may avoid knock-on damage and increase the contrast of weakly scattering objects. Scanning electron microscopes (SEMs) are well suited for low-energy electron microscopy with maximum electron energies of 30 keV, but they are mainly used for topography imaging of bulk samples. Implementation of a scanning transmission electron microscopy (STEM) detector and a charge-coupled-device camera for the acquisition of on-axis transmission electron diffraction (TED) patterns, in combination with recent resolution improvements, make SEMs highly interesting for structure analysis of some electron-transparent specimens which are traditionally investigated by TEM. A new aspect is correlative SEM, STEM, and TED imaging from the same specimen region in a SEM which leads to a wealth of information. Simultaneous image acquisition gives information on surface topography, inner structure including crystal defects and qualitative material contrast. Lattice-fringe resolution is obtained in bright-field STEM imaging. The benefits of correlative SEM/STEM/TED imaging in a SEM are exemplified by structure analyses from representative sample classes such as nanoparticulates and bulk materials.

A national facility for biological cryo-electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Saibil, Helen R., E-mail: h.saibil@mail.cryst.bbk.ac.uk; Grünewald, Kay; Stuart, David I.

2015-01-01

This review provides a brief update on the use of cryo-electron microscopy for integrated structural biology, along with an overview of the plans for the UK national facility for electron microscopy being built at the Diamond synchrotron. Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided ofmore » the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback.« less

Multi-pass transmission electron microscopy

DOE PAGES

Juffmann, Thomas; Koppell, Stewart A.; Klopfer, Brannon B.; ...

2017-05-10

Feynman once asked physicists to build better electron microscopes to be able to watch biology at work. While electron microscopes can now provide atomic resolution, electron beam induced specimen damage precludes high resolution imaging of sensitive materials, such as single proteins or polymers. Here, we use simulations to show that an electron microscope based on a multi-pass measurement protocol enables imaging of single proteins, without averaging structures over multiple images. While we demonstrate the method for particular imaging targets, the approach is broadly applicable and is expected to improve resolution and sensitivity for a range of electron microscopy imaging modalities,more » including, for example, scanning and spectroscopic techniques. The approach implements a quantum mechanically optimal strategy which under idealized conditions can be considered interaction-free.« less

High-Resolution of Electron Microscopy of Montmorillonite and Montmorillonite/Epoxy Nanocomposites

DTIC Science & Technology

2005-01-01

AFRL-ML-WP-TP-2006-464 HIGH-RESOLUTION OF ELECTRON MICROSCOPY OF MONTMORILLONITE AND MONTMORILLONITE /EPOXY NANOCOMPOSITES Lawrence F...HIGH-RESOLUTION OF ELECTRON MICROSCOPY OF MONTMORILLONITE AND MONTMORILLONITE /EPOXY NANOCOMPOSITES 5c. PROGRAM ELEMENT NUMBER 62102F 5d...transmission electron microscopy the structure and morphology of montmorillonite (MMT), a material of current interest for use in polymer nanocomposites, was

Transmission electron microscopy of amyloid fibrils.

PubMed

Gras, Sally L; Waddington, Lynne J; Goldie, Kenneth N

2011-01-01

Transmission Electron Microscopy of negatively stained and cryo-prepared specimens allows amyloid fibrils to be visualised at high resolution in a dried or a hydrated state, and is an essential method for characterising the morphology of fibrils and pre-fibrillar species. We outline the key steps involved in the preparation and observation of samples using negative staining and cryo-electron preservation. We also discuss methods to measure fibril characteristics, such as fibril width, from electron micrographs.

Near-infrared branding efficiently correlates light and electron microscopy.

PubMed

Bishop, Derron; Nikić, Ivana; Brinkoetter, Mary; Knecht, Sharmon; Potz, Stephanie; Kerschensteiner, Martin; Misgeld, Thomas

2011-06-05

The correlation of light and electron microscopy of complex tissues remains a major challenge. Here we report near-infrared branding (NIRB), which facilitates such correlation by using a pulsed, near-infrared laser to create defined fiducial marks in three dimensions in fixed tissue. As these marks are fluorescent and can be photo-oxidized to generate electron contrast, they can guide re-identification of previously imaged structures as small as dendritic spines by electron microscopy.

Application of environmental scanning electron microscopy to determine biological surface structure.

PubMed

Kirk, S E; Skepper, J N; Donald, A M

2009-02-01

The use of environmental scanning electron microscopy in biology is growing as more becomes understood about the advantages and limitations of the technique. These are discussed and we include new evidence about the effect of environmental scanning electron microscopy imaging on the viability of mammalian cells. We show that although specimen preparation for high-vacuum scanning electron microscopy introduces some artefacts, there are also challenges in the use of environmental scanning electron microscopy, particularly at higher resolutions. This suggests the two technologies are best used in combination. We have used human monocyte-derived macrophages as a test sample, imaging their complicated and delicate membrane ruffles and protrusions. We have also explored the possibility of using environmental scanning electron microscopy for dynamic experiments, finding that mammalian cells cannot be imaged and kept alive in the environmental scanning electron microscopy. The dehydration step in which the cell surface is exposed causes irreversible damage, probably via loss of membrane integrity during liquid removal in the specimen chamber. Therefore, mammalian cells should be imaged after fixation where possible to protect against damage as a result of chamber conditions.

Writing silica structures in liquid with scanning transmission electron microscopy.

PubMed

van de Put, Marcel W P; Carcouët, Camille C M C; Bomans, Paul H H; Friedrich, Heiner; de Jonge, Niels; Sommerdijk, Nico A J M

2015-02-04

Silica nanoparticles are imaged in solution with scanning transmission electron microscopy (STEM) using a liquid cell with silicon nitride (SiN) membrane windows. The STEM images reveal that silica structures are deposited in well-defined patches on the upper SiN membranes upon electron beam irradiation. The thickness of the deposits is linear with the applied electron dose. Scanning electron microscopy (SEM) and atomic force microscopy (AFM) demonstrate that the deposited patches are a result of the merging of the original 20 nm-diameter nanoparticles, and that the related surface roughness depends on the electron dose rate used. Using this approach, sub-micrometer scale structures are written on the SiN in liquid by controlling the electron exposure as function of the lateral position. © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Environmental transmission electron microscopy for catalyst materials using a spherical aberration corrector.

PubMed

Takeda, Seiji; Kuwauchi, Yasufumi; Yoshida, Hideto

2015-04-01

Atomic resolution has been obtained using environmental transmission electron microscopy (ETEM) by installing a spherical aberration corrector (Cs-corrector) on the objective lens. Simultaneously, the technology for controlling the environment around a specimen in ETEM has advanced significantly in the past decade. Quantification methodology has recently been established for deriving relevant experimental data in catalyst materials from substantial and systematic ETEM observation at the atomic scale. With this background, this paper summarizes aspects of the evolutional microscopy technique: necessary conditions for atomic resolution in ETEM; reduction of the scattering of electrons by the medium surrounding a specimen; and an environmental cell for structural imaging of a crystalline specimen. The high spatial resolution of a Cs-corrected ETEM is demonstrated for different observation conditions. After statistical analysis combined with numerical image analysis of ETEM data is briefly described, the recent applications of the Cs-corrected ETEM to catalyst materials are reviewed. For gold nanoparticulate catalysts, the structural information on the reaction sites and adsorption sites are deduced. For Pt nanoparticulate catalysts, ETEM studies elucidate the correlation between the catalytic activity and the morphology of the nanoparticles. These studies also reveal oxidation and reduction on the topmost Pt surface layer at the atomic scale. Finally, current issues and the future perspectives of Cs-corrected ETEM are summarized, including the reproducibility of ETEM observation data, the control of environments, the critical evaluation of electron irradiation effects, the full implementation of transmission electron microscopy technology in ETEM, and the safety issues for an ETEM laboratory. Copyright © 2014 Elsevier B.V. All rights reserved.

Application of high-angle annular dark field scanning transmission electron microscopy, scanning transmission electron microscopy-energy dispersive X-ray spectrometry, and energy-filtered transmission electron microscopy to the characterization of nanoparticles in the environment.

PubMed

Utsunomiya, Satoshi; Ewing, Rodney C

2003-02-15

A major challenge to the development of a fundamental understanding of transport and retardation mechanisms of trace metal contaminants (<10 ppm) is their identification and characterization at the nanoscale. Atomic-scale techniques, such as conventional transmission electron microscopy, although powerful, are limited by the extremely small amounts of material that are examined. However, recent advances in electron microscopy provide a number of new analytical techniques that expand its application in environmental studies, particularly those concerning heavy metals on airborne particulates or water-borne colloids. High-angle annular dark field scanning transmission electron microscopy (HAADF-STEM), STEM-energy-dispersive X-ray spectrometry (EDX), and energy-filtered TEM (EFTEM) can be effectively used to identify and characterize nanoparticles. The image contrast in HAADF-STEM is strongly correlated to the atomic mass: heavier elements contribute to brighter contrast. Gold nanocrystals in pyrite and uranium nanocrystals in atmospheric aerosols have been identified by HAADF-STEM and STEM-EDX mapping and subsequently characterized by high-resolution TEM (HRTEM). EFTEM was used to identify U and Fe nanocrystals embedded in an aluminosilicate. A rare, As-bearing nanophase, westerveldite (FeAs), was identified by STEM-EDX and HRTEM. The combined use of these techniques greatly expands the effective application of electron microscopy in environmental studies, especially when applied to metals of very low concentrations. This paper describes examples of how these electron microbeam techniques can be used in combination to characterize a low concentration of heavy metals (a few ppm) on nanoscale particles.

Attosecond electron pulse trains and quantum state reconstruction in ultrafast transmission electron microscopy

NASA Astrophysics Data System (ADS)

Priebe, Katharina E.; Rathje, Christopher; Yalunin, Sergey V.; Hohage, Thorsten; Feist, Armin; Schäfer, Sascha; Ropers, Claus

2017-12-01

Ultrafast electron and X-ray imaging and spectroscopy are the basis for an ongoing revolution in the understanding of dynamical atomic-scale processes in matter. The underlying technology relies heavily on laser science for the generation and characterization of ever shorter pulses. Recent findings suggest that ultrafast electron microscopy with attosecond-structured wavefunctions may be feasible. However, such future technologies call for means to both prepare and fully analyse the corresponding free-electron quantum states. Here, we introduce a framework for the preparation, coherent manipulation and characterization of free-electron quantum states, experimentally demonstrating attosecond electron pulse trains. Phase-locked optical fields coherently control the electron wavefunction along the beam direction. We establish a new variant of quantum state tomography—`SQUIRRELS'—for free-electron ensembles. The ability to tailor and quantitatively map electron quantum states will promote the nanoscale study of electron-matter entanglement and new forms of ultrafast electron microscopy down to the attosecond regime.

Relative merits and limiting factors for x-ray and electron microscopy of thick, hydrated organic materials.

PubMed

Du, Ming; Jacobsen, Chris

2018-01-01

Electron and x-ray microscopes allow one to image the entire, unlabeled structure of hydrated materials at a resolution well beyond what visible light microscopes can achieve. However, both approaches involve ionizing radiation, so that radiation damage must be considered as one of the limits to imaging. Drawing upon earlier work, we describe here a unified approach to estimating the image contrast (and thus the required exposure and corresponding radiation dose) in both x-ray and electron microscopy. This approach accounts for factors such as plural and inelastic scattering, and (in electron microscopy) the use of energy filters to obtain so-called "zero loss" images. As expected, it shows that electron microscopy offers lower dose for specimens thinner than about 1 µm (such as for studies of macromolecules, viruses, bacteria and archaebacteria, and thin sectioned material), while x-ray microscopy offers superior characteristics for imaging thicker specimen such as whole eukaryotic cells, thick-sectioned tissues, and organs. The required radiation dose scales strongly as a function of the desired spatial resolution, allowing one to understand the limits of live and frozen hydrated specimen imaging. Finally, we consider the factors limiting x-ray microscopy of thicker materials, suggesting that specimens as thick as a whole mouse brain can be imaged with x-ray microscopes without significant image degradation should appropriate image reconstruction methods be identified. Copyright © 2017 Elsevier B.V. All rights reserved.

Microscopy with slow electrons: from LEEM to XPEEM

ScienceCinema

Bauer, Ernst [Arizona State University, Phoenix, Arizona, United States

2017-12-09

The short penetration and escape depth of electrons with energies below 1 keV make them ideally suited for the study of surfaces and ultrathin films. The combination of the low energy electrons and the high lateral resolution of a microscope produces a powerful method for the characterization of nanostructures on bulk samples, in particular if the microscope is equipped with an imaging energy filter and connected to a synchrotron radiation source. Comprehensive characterization by imaging, diffraction, and spectroscope of the structural, chemical, and magnetic properties is then possible. The Talk will describe the various imaging techniques in using reflected and emitted electrons in low-energy electron microscopy (LEEM) and x-ray photoemission electron microscopy (XPEEM), with an emphasis on magnetic materials with spin-polarized LEEM and x-ray magnetic circular dichroism PEEM. The talk with end with an outlook on future possibilities.

New modes of electron microscopy for materials science enabled by fast direct electron detectors

NASA Astrophysics Data System (ADS)

Minor, Andrew

There is an ongoing revolution in the development of electron detector technology that has enabled modes of electron microscopy imaging that had only before been theorized. The age of electron microscopy as a tool for imaging is quickly giving way to a new frontier of multidimensional datasets to be mined. These improvements in electron detection have enabled cryo-electron microscopy to resolve the three-dimensional structures of non-crystalized proteins, revolutionizing structural biology. In the physical sciences direct electron detectors has enabled four-dimensional reciprocal space maps of materials at atomic resolution, providing all the structural information about nanoscale materials in one experiment. This talk will highlight the impact of direct electron detectors for materials science, including a new method of scanning nanobeam diffraction. With faster detectors we can take a series of 2D diffraction patterns at each position in a 2D STEM raster scan resulting in a four-dimensional data set. For thin film analysis, direct electron detectors hold the potential to enable strain, polarization, composition and electrical field mapping over relatively large fields of view, all from a single experiment.

Electron tomography of HEK293T cells using scanning electron microscope-based scanning transmission electron microscopy.

PubMed

You, Yun-Wen; Chang, Hsun-Yun; Liao, Hua-Yang; Kao, Wei-Lun; Yen, Guo-Ji; Chang, Chi-Jen; Tsai, Meng-Hung; Shyue, Jing-Jong

2012-10-01

Based on a scanning electron microscope operated at 30 kV with a homemade specimen holder and a multiangle solid-state detector behind the sample, low-kV scanning transmission electron microscopy (STEM) is presented with subsequent electron tomography for three-dimensional (3D) volume structure. Because of the low acceleration voltage, the stronger electron-atom scattering leads to a stronger contrast in the resulting image than standard TEM, especially for light elements. Furthermore, the low-kV STEM yields less radiation damage to the specimen, hence the structure can be preserved. In this work, two-dimensional STEM images of a 1-μm-thick cell section with projection angles between ±50° were collected, and the 3D volume structure was reconstructed using the simultaneous iterative reconstructive technique algorithm with the TomoJ plugin for ImageJ, which are both public domain software. Furthermore, the cross-sectional structure was obtained with the Volume Viewer plugin in ImageJ. Although the tilting angle is constrained and limits the resulting structural resolution, slicing the reconstructed volume generated the depth profile of the thick specimen with sufficient resolution to examine cellular uptake of Au nanoparticles, and the final position of these nanoparticles inside the cell was imaged.

Contributed review: Review of integrated correlative light and electron microscopy.

PubMed

Timmermans, F J; Otto, C

2015-01-01

New developments in the field of microscopy enable to acquire increasing amounts of information from large sample areas and at an increased resolution. Depending on the nature of the technique, the information may reveal morphological, structural, chemical, and still other sample characteristics. In research fields, such as cell biology and materials science, there is an increasing demand to correlate these individual levels of information and in this way to obtain a better understanding of sample preparation and specific sample properties. To address this need, integrated systems were developed that combine nanometer resolution electron microscopes with optical microscopes, which produce chemically or label specific information through spectroscopy. The complementary information from electron microscopy and light microscopy presents an opportunity to investigate a broad range of sample properties in a correlated fashion. An important part of correlating the differences in information lies in bridging the different resolution and image contrast features. The trend to analyse samples using multiple correlated microscopes has resulted in a new research field. Current research is focused, for instance, on (a) the investigation of samples with nanometer scale distribution of inorganic and organic materials, (b) live cell analysis combined with electron microscopy, and (c) in situ spectroscopic and electron microscopy analysis of catalytic materials, but more areas will benefit from integrated correlative microscopy.

Araldite as an Embedding Medium for Electron Microscopy

PubMed Central

Glauert, Audrey M.; Glauert, R. H.

1958-01-01

Epoxy resins are suitable media for embedding for electron microscopy, as they set uniformly with virtually no shrinkage. A mixture of araldite epoxy resins has been developed which is soluble in ethanol, and which yields a block of the required hardness for thin sectioning. The critical modifications to the conventional mixtures are the choice of a plasticized resin in conjunction with an aliphatic anhydride as the hardener. The hardness of the final block can be varied by incorporating additional plasticizer, and the rate of setting can be controlled by the use of an amine accelerator. The properties of the araldite mixture can be varied quite widely by adjusting the proportions of the various constituents. The procedure for embedding biological specimens is similar to that employed with methacrylates, although longer soaking times are recommended to ensure the complete penetration of the more viscous epoxy resin. An improvement in the preservation of the fine structure of a variety of specimens has already been reported, and a typical electron microgram illustrates the present paper. PMID:13525433

The application of scanning electron microscopy to fractography

DOE Office of Scientific and Technical Information (OSTI.GOV)

Brooks, C.R.; McGill, B.L.

1994-10-01

Many failures involve fracture, and determination of the fracture process is a key factor in understanding the failure. This is frequently accomplished by characterizing the topography of the fracture surface. Scanning electron microscopy has a prominent role in fractography due to three features of the scanning electron microscope (SEM): high resolution, great depth of field, and the ability to obtain chemical information via analysis of the X-rays generated by the electrons. A qualitative treatment is presented of the interaction of electrons with a sample and the effect of the SEM operating parameters on image formation, quality, and X-ray analysis. Fractographsmore » are presented to illustrate these features of scanning electron microscopy and to illustrate the limitations and precautions in obtaining fractographs and x-ray analyses. The review is concluded with examples of fracture surface features of metallic, ceramic, and polymeric materials.« less

SEGMENTATION OF MITOCHONDRIA IN ELECTRON MICROSCOPY IMAGES USING ALGEBRAIC CURVES.

PubMed

Seyedhosseini, Mojtaba; Ellisman, Mark H; Tasdizen, Tolga

2013-01-01

High-resolution microscopy techniques have been used to generate large volumes of data with enough details for understanding the complex structure of the nervous system. However, automatic techniques are required to segment cells and intracellular structures in these multi-terabyte datasets and make anatomical analysis possible on a large scale. We propose a fully automated method that exploits both shape information and regional statistics to segment irregularly shaped intracellular structures such as mitochondria in electron microscopy (EM) images. The main idea is to use algebraic curves to extract shape features together with texture features from image patches. Then, these powerful features are used to learn a random forest classifier, which can predict mitochondria locations precisely. Finally, the algebraic curves together with regional information are used to segment the mitochondria at the predicted locations. We demonstrate that our method outperforms the state-of-the-art algorithms in segmentation of mitochondria in EM images.

Correlative cryo-fluorescence light microscopy and cryo-electron tomography of Streptomyces.

PubMed

Koning, Roman I; Celler, Katherine; Willemse, Joost; Bos, Erik; van Wezel, Gilles P; Koster, Abraham J

2014-01-01

Light microscopy and electron microscopy are complementary techniques that in a correlative approach enable identification and targeting of fluorescently labeled structures in situ for three-dimensional imaging at nanometer resolution. Correlative imaging allows electron microscopic images to be positioned in a broader temporal and spatial context. We employed cryo-correlative light and electron microscopy (cryo-CLEM), combining cryo-fluorescence light microscopy and cryo-electron tomography, on vitrified Streptomyces bacteria to study cell division. Streptomycetes are mycelial bacteria that grow as long hyphae and reproduce via sporulation. On solid media, Streptomyces subsequently form distinct aerial mycelia where cell division leads to the formation of unigenomic spores which separate and disperse to form new colonies. In liquid media, only vegetative hyphae are present divided by noncell separating crosswalls. Their multicellular life style makes them exciting model systems for the study of bacterial development and cell division. Complex intracellular structures have been visualized with transmission electron microscopy. Here, we describe the methods for cryo-CLEM that we applied for studying Streptomyces. These methods include cell growth, fluorescent labeling, cryo-fixation by vitrification, cryo-light microscopy using a Linkam cryo-stage, image overlay and relocation, cryo-electron tomography using a Titan Krios, and tomographic reconstruction. Additionally, methods for segmentation, volume rendering, and visualization of the correlative data are described. © 2014 Elsevier Inc. All rights reserved.

Cryo-Scanning Electron Microscopy (SEM) and Scanning Transmission Electron Microscopy (STEM)-in-SEM for Bio- and Organo-Mineral Interface Characterization in the Environment.

PubMed

Wille, Guillaume; Hellal, Jennifer; Ollivier, Patrick; Richard, Annie; Burel, Agnes; Jolly, Louis; Crampon, Marc; Michel, Caroline

2017-12-01

Understanding biofilm interactions with surrounding substratum and pollutants/particles can benefit from the application of existing microscopy tools. Using the example of biofilm interactions with zero-valent iron nanoparticles (nZVI), this study aims to apply various approaches in biofilm preparation and labeling for fluorescent or electron microscopy and energy dispersive X-ray spectrometry (EDS) microanalysis for accurate observations. According to the targeted microscopy method, biofilms were sampled as flocs or attached biofilm, submitted to labeling using 4',6-diamidino-2-phenylindol, lectins PNA and ConA coupled to fluorescent dye or gold nanoparticles, and prepared for observation (fixation, cross-section, freezing, ultramicrotomy). Fluorescent microscopy revealed that nZVI were embedded in the biofilm structure as aggregates but the resolution was insufficient to observe individual nZVI. Cryo-scanning electron microscopy (SEM) observations showed nZVI aggregates close to bacteria, but it was not possible to confirm direct interactions between nZVI and cell membranes. Scanning transmission electron microscopy in the SEM (STEM-in-SEM) showed that nZVI aggregates could enter the biofilm to a depth of 7-11 µm. Bacteria were surrounded by a ring of extracellular polymeric substances (EPS) preventing direct nZVI/membrane interactions. STEM/EDS mapping revealed a co-localization of nZVI aggregates with lectins suggesting a potential role of EPS in nZVI embedding. Thus, the combination of divergent microscopy approaches is a good approach to better understand and characterize biofilm/metal interactions.

4D multiple-cathode ultrafast electron microscopy

PubMed Central

Baskin, John Spencer; Liu, Haihua; Zewail, Ahmed H.

2014-01-01

Four-dimensional multiple-cathode ultrafast electron microscopy is developed to enable the capture of multiple images at ultrashort time intervals for a single microscopic dynamic process. The dynamic process is initiated in the specimen by one femtosecond light pulse and probed by multiple packets of electrons generated by one UV laser pulse impinging on multiple, spatially distinct, cathode surfaces. Each packet is distinctly recorded, with timing and detector location controlled by the cathode configuration. In the first demonstration, two packets of electrons on each image frame (of the CCD) probe different times, separated by 19 picoseconds, in the evolution of the diffraction of a gold film following femtosecond heating. Future elaborations of this concept to extend its capabilities and expand the range of applications of 4D ultrafast electron microscopy are discussed. The proof-of-principle demonstration reported here provides a path toward the imaging of irreversible ultrafast phenomena of materials, and opens the door to studies involving the single-frame capture of ultrafast dynamics using single-pump/multiple-probe, embedded stroboscopic imaging. PMID:25006261

4D multiple-cathode ultrafast electron microscopy.

PubMed

Baskin, John Spencer; Liu, Haihua; Zewail, Ahmed H

2014-07-22

Four-dimensional multiple-cathode ultrafast electron microscopy is developed to enable the capture of multiple images at ultrashort time intervals for a single microscopic dynamic process. The dynamic process is initiated in the specimen by one femtosecond light pulse and probed by multiple packets of electrons generated by one UV laser pulse impinging on multiple, spatially distinct, cathode surfaces. Each packet is distinctly recorded, with timing and detector location controlled by the cathode configuration. In the first demonstration, two packets of electrons on each image frame (of the CCD) probe different times, separated by 19 picoseconds, in the evolution of the diffraction of a gold film following femtosecond heating. Future elaborations of this concept to extend its capabilities and expand the range of applications of 4D ultrafast electron microscopy are discussed. The proof-of-principle demonstration reported here provides a path toward the imaging of irreversible ultrafast phenomena of materials, and opens the door to studies involving the single-frame capture of ultrafast dynamics using single-pump/multiple-probe, embedded stroboscopic imaging.

Experiments in electron microscopy: from metals to nerves

NASA Astrophysics Data System (ADS)

Unwin, Nigel

2015-04-01

Electron microscopy has advanced remarkably as a tool for biological structure research since the development of methods to examine radiation-sensitive unstained specimens and the introduction of cryo-techniques. Structures of biological molecules at near-atomic resolution can now be obtained from images of single particles as well as crystalline arrays. It has also become possible to analyze structures of molecules in their functional context, i.e. in their natural membrane or cellular setting, and in an ionic environment like that in living tissue. Electron microscopy is thus opening ways to answer definitively questions about physiological mechanisms. Here I recall a number of experiments contributing to, and benefiting from the technical advances that have taken place. I begin—in the spirit of this crystallography series—with some biographical background, and then sketch the path to an analysis by time-resolved microscopy of the opening mechanism of an ion channel (nicotinic acetylcholine receptor). This analysis illustrates how electron imaging can be combined with freeze-trapping to illuminate a transient biological event: in our case, chemical-to-electrical transduction at the nerve-muscle synapse.

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy.

PubMed

Hermannsdörfer, Justus; de Jonge, Niels

2017-02-05

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline.

Studying Dynamic Processes of Nano-sized Objects in Liquid using Scanning Transmission Electron Microscopy

PubMed Central

Hermannsdörfer, Justus; de Jonge, Niels

2017-01-01

Samples fully embedded in liquid can be studied at a nanoscale spatial resolution with Scanning Transmission Electron Microscopy (STEM) using a microfluidic chamber assembled in the specimen holder for Transmission Electron Microscopy (TEM) and STEM. The microfluidic system consists of two silicon microchips supporting thin Silicon Nitride (SiN) membrane windows. This article describes the basic steps of sample loading and data acquisition. Most important of all is to ensure that the liquid compartment is correctly assembled, thus providing a thin liquid layer and a vacuum seal. This protocol also includes a number of tests necessary to perform during sample loading in order to ensure correct assembly. Once the sample is loaded in the electron microscope, the liquid thickness needs to be measured. Incorrect assembly may result in a too-thick liquid, while a too-thin liquid may indicate the absence of liquid, such as when a bubble is formed. Finally, the protocol explains how images are taken and how dynamic processes can be studied. A sample containing AuNPs is imaged both in pure water and in saline. PMID:28190028

Serial block face scanning electron microscopy--the future of cell ultrastructure imaging.

PubMed

Hughes, Louise; Hawes, Chris; Monteith, Sandy; Vaughan, Sue

2014-03-01

One of the major drawbacks in transmission electron microscopy has been the production of three-dimensional views of cells and tissues. Currently, there is no one suitable 3D microscopy technique that answers all questions and serial block face scanning electron microscopy (SEM) fills the gap between 3D imaging using high-end fluorescence microscopy and the high resolution offered by electron tomography. In this review, we discuss the potential of the serial block face SEM technique for studying the three-dimensional organisation of animal, plant and microbial cells.

Quantitative Cryo-Scanning Transmission Electron Microscopy of Biological Materials.

PubMed

Elbaum, Michael

2018-05-11

Electron tomography provides a detailed view into the 3D structure of biological cells and tissues. Physical fixation by vitrification of the aqueous medium provides the most faithful preservation of biological specimens in the native, fully hydrated state. Cryo-microscopy is challenging, however, because of the sensitivity to electron irradiation and due to the weak electron scattering of organic material. Tomography is even more challenging because of the dependence on multiple exposures of the same area. Tomographic imaging is typically performed in wide-field transmission electron microscopy (TEM) mode with phase contrast generated by defocus. Scanning transmission electron microscopy (STEM) is an alternative mode based on detection of scattering from a focused probe beam, without imaging optics following the specimen. While careful configuration of the illumination and detectors is required to generate useful contrast, STEM circumvents the major restrictions of phase contrast TEM to very thin specimens and provides a signal that is more simply interpreted in terms of local composition and density. STEM has gained popularity in recent years for materials science. The extension of STEM to cryomicroscopy and tomography of cells and macromolecules is summarized herein. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

4D electron microscopy: principles and applications.

PubMed

Flannigan, David J; Zewail, Ahmed H

2012-10-16

The transmission electron microscope (TEM) is a powerful tool enabling the visualization of atoms with length scales smaller than the Bohr radius at a factor of only 20 larger than the relativistic electron wavelength of 2.5 pm at 200 keV. The ability to visualize matter at these scales in a TEM is largely due to the efforts made in correcting for the imperfections in the lens systems which introduce aberrations and ultimately limit the achievable spatial resolution. In addition to the progress made in increasing the spatial resolution, the TEM has become an all-in-one characterization tool. Indeed, most of the properties of a material can be directly mapped in the TEM, including the composition, structure, bonding, morphology, and defects. The scope of applications spans essentially all of the physical sciences and includes biology. Until recently, however, high resolution visualization of structural changes occurring on sub-millisecond time scales was not possible. In order to reach the ultrashort temporal domain within which fundamental atomic motions take place, while simultaneously retaining high spatial resolution, an entirely new approach from that of millisecond-limited TEM cameras had to be conceived. As shown below, the approach is also different from that of nanosecond-limited TEM, whose resolution cannot offer the ultrafast regimes of dynamics. For this reason "ultrafast electron microscopy" is reserved for the field which is concerned with femtosecond to picosecond resolution capability of structural dynamics. In conventional TEMs, electrons are produced by heating a source or by applying a strong extraction field. Both methods result in the stochastic emission of electrons, with no control over temporal spacing or relative arrival time at the specimen. The timing issue can be overcome by exploiting the photoelectric effect and using pulsed lasers to generate precisely timed electron packets of ultrashort duration. The spatial and temporal resolutions

Frontiers of in situ electron microscopy

DOE PAGES

Zheng, Haimei; Zhu, Yimei; Meng, Shirley Ying

2015-01-01

In situ transmission electron microscopy (TEM) has become an increasingly important tool for materials characterization. It provides key information on the structural dynamics of a material during transformations and the correlation between structure and properties of materials. With the recent advances in instrumentation, including aberration corrected optics, sample environment control, the sample stage, and fast and sensitive data acquisition, in situ TEM characterization has become more and more powerful. In this article, a brief review of the current status and future opportunities of in situ TEM is included. It also provides an introduction to the six articles covered by inmore » this issue of MRS Bulletin explore the frontiers of in situ electron microscopy, including liquid and gas environmental TEM, dynamic four-dimensional TEM, nanomechanics, ferroelectric domain switching studied by in situ TEM, and state-of-the-art atomic imaging of light elements (i.e., carbon atoms) and individual defects.« less

Neurite density from magnetic resonance diffusion measurements at ultrahigh field: Comparison with light microscopy and electron microscopy

PubMed Central

Jespersen, Sune N.; Bjarkam, Carsten R.; Nyengaard, Jens R.; Chakravarty, M. Mallar; Hansen, Brian; Vosegaard, Thomas; Østergaard, Leif; Yablonskiy, Dmitriy; Nielsen, Niels Chr.; Vestergaard-Poulsen, Peter

2010-01-01

Due to its unique sensitivity to tissue microstructure, diffusion-weighted magnetic resonance imaging (MRI) has found many applications in clinical and fundamental science. With few exceptions, a more precise correspondence between physiological or biophysical properties and the obtained diffusion parameters remain uncertain due to lack of specificity. In this work, we address this problem by comparing diffusion parameters of a recently introduced model for water diffusion in brain matter to light microscopy and quantitative electron microscopy. Specifically, we compare diffusion model predictions of neurite density in rats to optical myelin staining intensity and stereological estimation of neurite volume fraction using electron microscopy. We find that the diffusion model describes data better and that its parameters show stronger correlation with optical and electron microscopy, and thus reflect myelinated neurite density better than the more frequently used diffusion tensor imaging (DTI) and cumulant expansion methods. Furthermore, the estimated neurite orientations capture dendritic architecture more faithfully than DTI diffusion ellipsoids. PMID:19732836

Electron microscopy of intermediate filaments: teaming up with atomic force and confocal laser scanning microscopy.

PubMed

Kreplak, Laurent; Richter, Karsten; Aebi, Ueli; Herrmann, Harald

2008-01-01

Intermediate filaments (IFs) were originally discovered and defined by electron microscopy in myoblasts. In the following it was demonstrated and confirmed that they constitute, in addition to microtubules and microfilaments, a third independent, general filament system in the cytoplasm of most metazoan cells. In contrast to the other two systems, IFs are present in cells in two principally distinct cytoskeletal forms: (i) extended and free-running filament arrays in the cytoplasm that are integrated into the cytoskeleton by associated proteins of the plakin type; and (ii) a membrane- and chromatin-bound thin 'lamina' of a more or less regular network of interconnected filaments made from nuclear IF proteins, the lamins, which differ in several important structural aspects from cytoplasmic IF proteins. In man, more than 65 genes code for distinct IF proteins that are expressed during embryogenesis in various routes of differentiation in a tightly controlled manner. IF proteins exhibit rather limited sequence identity implying that the different types of IFs have distinct biochemical properties. Hence, to characterize the structural properties of the various IFs, in vitro assembly regimes have been developed in combination with different visualization methods such as transmission electron microscopy of fixed and negatively stained samples as well as methods that do not use staining such as scanning transmission electron microscopy (STEM) and cryoelectron microscopy as well as atomic force microscopy. Moreover, with the generation of both IF-type specific antibodies and chimeras of fluorescent proteins and IF proteins, it has become possible to investigate the subcellular organization of IFs by correlative fluorescence and electron microscopic methods. The combination of these powerful methods should help to further develop our understanding of nuclear architecture, in particular how nuclear subcompartments are organized and in which way lamins are involved.

Simulation of Mirror Electron Microscopy Caustic Images in Three-Dimensions

NASA Astrophysics Data System (ADS)

Kennedy, S. M.; Zheng, C. X.; Jesson, D. E.

A full, three-dimensional (3D) ray tracing approach is developed to simulate the caustics visible in mirror electron microscopy (MEM). The method reproduces MEM image contrast resulting from 3D surface relief. To illustrate the potential of the simulation methods, we study the evolution of crater contrast associated with a movie of GaAs structures generated by the droplet epitaxy technique. Specifically, we simulate the image contrast resulting from both a precursor stage and the final crater morphology which is consistent with an inverted pyramid consisting of (111) facet walls. The method therefore facilities the study of how self-assembled quantum structures evolve with time and, in particular, the development of anisotropic features including faceting.

Microcapsules on Streptococcus mutans serotypes by electron microscopy.

PubMed

Grenier, E M; Gray, R H; Loesche, W J; Eveland, W C

1977-02-01

Extracellular microcapsules have been demonstrated on cells of most serotypes of Streptococcus mutans by electron microscopy, using bacterial strains of the various serotypes and peroxidase labeled or unlabeled immune serum. A correlation was noted between the amount of capsular substance on the strains of S mutans examined and degree of antigenicity as expressed by the indirect fluorescent antibody (FA) title. A serotype d strain was shown to lose both antigenicity as determined by the FA reaction and capsular material as seen by electron microscopy with repeated in vitro passage. When 10% unheated rabbit serum was added to the medium, antigenicity and capsular material were restored.

Environmental scanning electron microscopy gold immunolabeling in cell biology.

PubMed

Rosso, Francesco; Papale, Ferdinando; Barbarisi, Alfonso

2013-01-01

Immunogold labeling (IGL) technique has been utilized by many authors in combination with scanning electron microscopy (SEM) and transmission electron microscopy (TEM) to obtain the identification/localization of receptors and antigens, both in cells and tissues. Environmental scanning electron microscopy (ESEM) represents an important tool in biomedical research, since it does not require any severe processing of the sample, lowering the risk of generating artifacts and interfere with the IGL procedure. The absence of metal coating could yield further advantages for our purpose as the labeling detection is based on the atomic number difference between nanogold spheres and the biological material. Using the gaseous secondary electron detector, compositional contrast is easily revealed by the backscattered electron component of the signal. In spite of this fact, only few published papers present a combination of ESEM and IGL. Hereby we present our method, optimized to improve the intensity and the specificity of the labeling signal, in order to obtain a semiquantitative evaluation of the labeling signal.In particular, we used a combination of IGL and ESEM to detect the presence of a protein on the cell surface. To achieve this purpose, we chose as an experimental system 3T3 Swiss albino mouse fibroblasts and galectin-3.

HALE STAIN FOR SIALIC ACID-CONTAINING MUCINS. ADAPTATION TO ELECTRON MICROSCOPY.

PubMed

GASIC, G; BERWICK, L

1963-10-01

The feasibility of using the Hale stain to identify cellular sialic acid-containing mucins by electron microscopy was investigated. Three kinds of mouse ascites tumor cells were fixed in neutral buffered formalin, exposed to fresh colloidal ferric oxide, treated with potassium ferrocyanide, imbedded in Selectron, and sectioned for electron microscopy. Additional staining with uranyl acetate and potassium permanganate was done after sectioning in order to increase contrast. Those cells known to be coated with sialomucin showed deposits of electron-opaque ferric ferrocyanide crystals in the areas where sialomucin concentrations were expected. When these cells were treated with neuraminidase beforehand, these deposits did not appear. It was concluded that, with the precautions and modifications described, the Hale stain can be successfully combined with electron microscopy to identify sialomucin.

Three-Dimensional Electron Microscopy Simulation with the CASINO Monte Carlo Software

PubMed Central

Demers, Hendrix; Poirier-Demers, Nicolas; Couture, Alexandre Réal; Joly, Dany; Guilmain, Marc; de Jonge, Niels; Drouin, Dominique

2011-01-01

Monte Carlo softwares are widely used to understand the capabilities of electron microscopes. To study more realistic applications with complex samples, 3D Monte Carlo softwares are needed. In this paper, the development of the 3D version of CASINO is presented. The software feature a graphical user interface, an efficient (in relation to simulation time and memory use) 3D simulation model, accurate physic models for electron microscopy applications, and it is available freely to the scientific community at this website: www.gel.usherbrooke.ca/casino/index.html. It can be used to model backscattered, secondary, and transmitted electron signals as well as absorbed energy. The software features like scan points and shot noise allow the simulation and study of realistic experimental conditions. This software has an improved energy range for scanning electron microscopy and scanning transmission electron microscopy applications. PMID:21769885

A simple energy filter for low energy electron microscopy/photoelectron emission microscopy instruments.

PubMed

Tromp, R M; Fujikawa, Y; Hannon, J B; Ellis, A W; Berghaus, A; Schaff, O

2009-08-05

Addition of an electron energy filter to low energy electron microscopy (LEEM) and photoelectron emission microscopy (PEEM) instruments greatly improves their analytical capabilities. However, such filters tend to be quite complex, both electron optically and mechanically. Here we describe a simple energy filter for the existing IBM LEEM/PEEM instrument, which is realized by adding a single scanning aperture slit to the objective transfer optics, without any further modifications to the microscope. This energy filter displays a very high energy resolution ΔE/E = 2 × 10(-5), and a non-isochromaticity of ∼0.5 eV/10 µm. The setup is capable of recording selected area electron energy spectra and angular distributions at 0.15 eV energy resolution, as well as energy filtered images with a 1.5 eV energy pass band at an estimated spatial resolution of ∼10 nm. We demonstrate the use of this energy filter in imaging and spectroscopy of surfaces using a laboratory-based He I (21.2 eV) light source, as well as imaging of Ag nanowires on Si(001) using the 4 eV energy loss Ag plasmon.

Evaluations of carbon nanotube field emitters for electron microscopy

NASA Astrophysics Data System (ADS)

Nakahara, Hitoshi; Kusano, Yoshikazu; Kono, Takumi; Saito, Yahachi

2009-11-01

Brightness of carbon nanotube (CNT) emitters was already reported elsewhere. However, brightness of electron emitter is affected by a virtual source size of the emitter, which strongly depends on electron optical configuration around the emitter. In this work, I- V characteristics and brightness of a CNT emitter are measured under a practical field emission electron gun (e-gun) configuration to investigate availability of CNT for electron microscopy. As a result, it is obtained that an emission area of MWNT is smaller than its tip surface area, and the emission area corresponds to a five-membered-ring with 2nd nearest six-membered-rings on the MWNT cap surface. Reduced brightness of MWNT is measured as at least 2.6×109 A/m 2 sr V. It is concluded that even a thick MWNT has enough brightness under a practical e-gun electrode configuration and suitable for electron microscopy.

The Effect of Electron Beam Irradiation in Environmental Scanning Transmission Electron Microscopy of Whole Cells in Liquid.

PubMed

Hermannsdörfer, Justus; Tinnemann, Verena; Peckys, Diana B; de Jonge, Niels

2016-06-01

Whole cells can be studied in their native liquid environment using electron microscopy, and unique information about the locations and stoichiometry of individual membrane proteins can be obtained from many cells thus taking cell heterogeneity into account. Of key importance for the further development of this microscopy technology is knowledge about the effect of electron beam radiation on the samples under investigation. We used environmental scanning electron microscopy (ESEM) with scanning transmission electron microscopy (STEM) detection to examine the effect of radiation for whole fixed COS7 fibroblasts in liquid. The main observation was the localization of nanoparticle labels attached to epidermal growth factor receptors (EGFRs). It was found that the relative distances between the labels remained mostly unchanged (<1.5%) for electron doses ranging from the undamaged native state at 10 e-/Å2 toward 103 e-/Å2. This dose range was sufficient to determine the EGFR locations with nanometer resolution and to distinguish between monomers and dimers. Various different forms of radiation damage became visible at higher doses, including severe dislocation, and the dissolution of labels.

Three-dimensional electron microscopy simulation with the CASINO Monte Carlo software.

PubMed

Demers, Hendrix; Poirier-Demers, Nicolas; Couture, Alexandre Réal; Joly, Dany; Guilmain, Marc; de Jonge, Niels; Drouin, Dominique

2011-01-01

Monte Carlo softwares are widely used to understand the capabilities of electron microscopes. To study more realistic applications with complex samples, 3D Monte Carlo softwares are needed. In this article, the development of the 3D version of CASINO is presented. The software feature a graphical user interface, an efficient (in relation to simulation time and memory use) 3D simulation model, accurate physic models for electron microscopy applications, and it is available freely to the scientific community at this website: www.gel.usherbrooke.ca/casino/index.html. It can be used to model backscattered, secondary, and transmitted electron signals as well as absorbed energy. The software features like scan points and shot noise allow the simulation and study of realistic experimental conditions. This software has an improved energy range for scanning electron microscopy and scanning transmission electron microscopy applications. Copyright © 2011 Wiley Periodicals, Inc.

An overview of state-of-the-art image restoration in electron microscopy.

PubMed

Roels, J; Aelterman, J; Luong, H Q; Lippens, S; Pižurica, A; Saeys, Y; Philips, W

2018-06-08

In Life Science research, electron microscopy (EM) is an essential tool for morphological analysis at the subcellular level as it allows for visualization at nanometer resolution. However, electron micrographs contain image degradations such as noise and blur caused by electromagnetic interference, electron counting errors, magnetic lens imperfections, electron diffraction, etc. These imperfections in raw image quality are inevitable and hamper subsequent image analysis and visualization. In an effort to mitigate these artefacts, many electron microscopy image restoration algorithms have been proposed in the last years. Most of these methods rely on generic assumptions on the image or degradations and are therefore outperformed by advanced methods that are based on more accurate models. Ideally, a method will accurately model the specific degradations that fit the physical acquisition settings. In this overview paper, we discuss different electron microscopy image degradation solutions and demonstrate that dedicated artefact regularisation results in higher quality restoration and is applicable through recently developed probabilistic methods. © 2018 The Authors Journal of Microscopy © 2018 Royal Microscopical Society.

The New Electron Microscopy: Cells and Molecules in Three Dimensions | Poster

Cancer.gov

NCI recently announced the launch of the new National Cryo-Electron Microscopy Facility (NCEF) at the Frederick National Laboratory for Cancer Research (FNLCR). The launch comes while cryo-electron microscopy (cryo-EM) is enjoying the spotlight as a newly emerging, rapidly evolving technology with the potential to revolutionize the field of structural biology. Read more...

Imaging plasmodesmata with high-resolution scanning electron microscopy.

PubMed

Barton, Deborah A; Overall, Robyn L

2015-01-01

High-resolution scanning electron microscopy (HRSEM) is an effective tool to investigate the distribution of plasmodesmata within plant cell walls as well as to probe their complex, three-dimensional architecture. It is a useful alternative to traditional transmission electron microscopy (TEM) in which plasmodesmata are sectioned to reveal their internal substructures. Benefits of adopting an HRSEM approach to studies of plasmodesmata are that the specimen preparation methods are less complex and time consuming than for TEM, many plasmodesmata within a large region of tissue can be imaged in a single session, and three-dimensional information is readily available without the need for reconstructing TEM serial sections or employing transmission electron tomography, both of which are lengthy processes. Here we describe methods to prepare plant samples for HRSEM using pre- or postfixation extraction of cellular material in order to visualize plasmodesmata embedded within plant cell walls.

An inexpensive approach for bright-field and dark-field imaging by scanning transmission electron microscopy in scanning electron microscopy.

PubMed

Patel, Binay; Watanabe, Masashi

2014-02-01

Scanning transmission electron microscopy in scanning electron microscopy (STEM-in-SEM) is a convenient technique for soft materials characterization. Various specimen-holder geometries and detector arrangements have been used for bright-field (BF) STEM-in-SEM imaging. In this study, to further the characterization potential of STEM-IN-SEM, a new specimen holder has been developed to facilitate direct detection of BF signals and indirect detection of dark-field (DF) signals without the need for substantial instrument modification. DF imaging is conducted with the use of a gold (Au)-coated copper (Cu) plate attached to the specimen holder which directs highly scattered transmitted electrons to an off-axis yttrium-aluminum-garnet (YAG) detector. A hole in the copper plate allows for BF imaging with a transmission electron (TE) detector. The inclusion of an Au-coated Cu plate enhanced DF signal intensity. Experiments validating the acquisition of true DF signals revealed that atomic number (Z) contrast may be achieved for materials with large lattice spacing. However, materials with small lattice spacing still exhibit diffraction contrast effects in this approach. The calculated theoretical fine probe size is 1.8 nm. At 30 kV, in this indirect approach, DF spatial resolution is limited to 3.2 nm as confirmed experimentally.

Ultra low signals in ballistic electron emission microscopy

NASA Astrophysics Data System (ADS)

Heller, Eric

The extension of Scanning Tunneling Microscopy known as Ballistic Electron Emission Microscopy (BEEM) was expanded to allow useful data collection at lower signal levels than previously possible, and a critical BEEM shortcoming was discovered and quantified. As a separate effort, a new method for measuring SB-type step energies on Si(001) SA-type steps that under some circumstances is more accurate than previous methods was used and will be presented. Finally, extensive modifications to a Scanning Tunneling Microscope used for most of this research will be presented. First, it will be shown theoretically and experimentally that by amplifying the hot BEEM electrons that make up the useful BEEM signal before they are thermalized, internal gain can be applied specifically to these electrons without amplifying standard BEEM noise sources. It will be shown that BEEM with single hot electron sensitivity (approximately a factor of 1000 improvement in the minimum detectable BEEM signal) is attainable with modified commercially existing avalanche photodiodes. With this new low-signal capability, it was obvious that a new BEEM-like signal was being detected. We have discovered that photons generated by STM tunneling will create a false signal in most BEEM samples. Furthermore, we have characterized this effect which we call "STM-PC" and it will be demonstrated with Pd/SiO2/Si and Au/SiO2/Si samples that this false signal closely mimics BEEM and is easily confused for BEEM. We will discuss ways to separate real BEEM from this new effect. Separately, thermally generated kinks on A-type steps on the Si(001) surface were counted and analyzed to find the SB-type step energy. Previous work by others was extended by counting a new type of feature, the "switch" kink, to allow a more accurate determination of the energy of SB-steps in the presence of defects that can bow steps and cause non-thermal kinks. Considerable data collection along with this new extension allowed a more

Correlative Fluorescence and Electron Microscopy in 3D-Scanning Electron Microscope Perspective.

PubMed

Franks, Jonathan; Wallace, Callen T; Shibata, Masateru; Suga, Mitsuo; Erdman, Natasha; Stolz, Donna B; Watkins, Simon C

2017-04-03

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. Copyright © 2017 John Wiley & Sons, Inc.

In situ transmission electron microscopy of transistor operation and failure.

PubMed

Wang, Baoming; Islam, Zahabul; Haque, Aman; Chabak, Kelson; Snure, Michael; Heller, Eric; Glavin, Nicholas

2018-08-03

Microscopy is typically used as a post-mortem analytical tool in performance and reliability studies on nanoscale materials and devices. In this study, we demonstrate real time microscopy of the operation and failure of AlGaN/GaN high electron mobility transistors inside the transmission electron microscope. Loading until failure was performed on the electron transparent transistors to visualize the failure mechanisms caused by self-heating. At lower drain voltages, thermo-mechanical stresses induce irreversible microstructural deformation, mostly along the AlGaN/GaN interface, to initiate the damage process. At higher biasing, the self-heating deteriorates the gate and catastrophic failure takes place through metal/semiconductor inter-diffusion and/or buffer layer breakdown. This study indicates that the current trend of recreating the events, from damage nucleation to catastrophic failure, can be replaced by in situ microscopy for a quick and accurate account of the failure mechanisms.

Microfabricated high-bandpass foucault aperture for electron microscopy

DOEpatents

Glaeser, Robert; Cambie, Rossana; Jin, Jian

2014-08-26

A variant of the Foucault (knife-edge) aperture is disclosed that is designed to provide single-sideband (SSB) contrast at low spatial frequencies but retain conventional double-sideband (DSB) contrast at high spatial frequencies in transmission electron microscopy. The aperture includes a plate with an inner open area, a support extending from the plate at an edge of the open area, a half-circle feature mounted on the support and located at the center of the aperture open area. The radius of the half-circle portion of reciprocal space that is blocked by the aperture can be varied to suit the needs of electron microscopy investigation. The aperture is fabricated from conductive material which is preferably non-oxidizing, such as gold, for example.

Mechanisms of decoherence in electron microscopy.

PubMed

Howie, A

2011-06-01

The understanding and where possible the minimisation of decoherence mechanisms in electron microscopy were first studied in plasmon loss, diffraction contrast images but are of even more acute relevance in high resolution TEM phase contrast imaging and electron holography. With the development of phase retrieval techniques they merit further attention particularly when their effect cannot be eliminated by currently available energy filters. The roles of electronic excitation, thermal diffuse scattering, transition radiation and bremsstrahlung are examined here not only in the specimen but also in the electron optical column. Terahertz-range aloof beam electronic excitation appears to account satisfactorily for recent observations of decoherence in electron holography. An apparent low frequency divergence can emerge for the calculated classical bremsstrahlung event probability but can be ignored for photon wavelengths exceeding the required coherence distance or path lengths in the equipment. Most bremsstrahlung event probabilities are negligibly important except possibly in large-angle bending magnets or mandolin systems. A more reliable procedure for subtracting thermal diffuse scattering from diffraction pattern intensities is proposed. Copyright © 2010 Elsevier B.V. All rights reserved.

Transmission electron microscopy artifacts in characterization of the nanomaterial-cell interactions.

PubMed

Leung, Yu Hang; Guo, Mu Yao; Ma, Angel P Y; Ng, Alan M C; Djurišić, Aleksandra B; Degger, Natalie; Leung, Frederick C C

2017-07-01

We investigated transmission electron microscopy artifacts obtained using standard sample preparation protocols applied to the investigation of Escherichia coli cells exposed to common nanomaterials, such as TiO 2 , Ag, ZnO, and MgO. While the common protocols for some nanomaterials result only in known issues of nanomaterial-independent generation of anomalous deposits due to fixation and staining, for others, there are reactions between the nanomaterial and chemicals used for post-fixation or staining. Only in the case of TiO 2 do we observe only the known issues of nanomaterial-independent generation of anomalous deposits due to exceptional chemical stability of this material. For the other three nanomaterials, different artifacts are observed. For each of those, we identify causes of the observed problems and suggest alternative sample preparation protocols to avoid artifacts arising from the sample preparation, which is essential for correct interpretation of the obtained images and drawing correct conclusions on cell-nanomaterial interactions. Finally, we propose modified sample preparation and characterization protocols for comprehensive and conclusive investigations of nanomaterial-cell interactions using electron microscopy and for obtaining clear and unambiguous revelation whether the nanomaterials studied penetrate the cells or accumulate at the cell membranes. In only the case of MgO and ZnO, the unambiguous presence of Zn and Mg could be observed inside the cells.

Scanning electron microscopy of cells and tissues under fully hydrated conditions

PubMed Central

Thiberge, Stephan; Nechushtan, Amotz; Sprinzak, David; Gileadi, Opher; Behar, Vered; Zik, Ory; Chowers, Yehuda; Michaeli, Shulamit; Schlessinger, Joseph; Moses, Elisha

2004-01-01

A capability for scanning electron microscopy of wet biological specimens is presented. A membrane that is transparent to electrons protects the fully hydrated sample from the vacuum. The result is a hybrid technique combining the ease of use and ability to see into cells of optical microscopy with the higher resolution of electron microscopy. The resolution of low-contrast materials is ≈100 nm, whereas in high-contrast materials the resolution can reach 10 nm. Standard immunogold techniques and heavy-metal stains can be applied and viewed in the fluid to improve the contrast. Images present a striking combination of whole-cell morphology with a wealth of internal details. A possibility for direct inspection of tissue slices transpires, imaging only the external layer of cells. Simultaneous imaging with photons excited by the electrons incorporates data on material distribution, indicating a potential for multilabeling and specific scintillating markers. PMID:14988502

Atomic-scale mapping of electronic structures across heterointerfaces by cross-sectional scanning tunneling microscopy

NASA Astrophysics Data System (ADS)

Chiu, Ya-Ping; Huang, Bo-Chao; Shih, Min-Chuan; Huang, Po-Cheng; Chen, Chun-Wei

2015-09-01

Interfacial science has received much attention recently based on the development of state-of-the-art analytical tools that can create and manipulate the charge, spin, orbital, and lattice degrees of freedom at interfaces. Motivated by the importance of nanoscale interfacial science that governs device operation, we present a technique to probe the electronic characteristics of heterointerfaces with atomic resolution. In this work, the interfacial characteristics of heteroepitaxial structures are investigated and the fundamental mechanisms that pertain in these systems are elucidated through cross-sectional scanning tunneling microscopy (XSTM). The XSTM technique is employed here to directly observe epitaxial interfacial structures and probe local electronic properties with atomic-level capability. Scanning tunneling microscopy and spectroscopy experiments with atomic precision provide insight into the origin and spatial distribution of electronic properties across heterointerfaces. The first part of this report provides a brief description of the cleavage technique and spectroscopy analysis in XSTM measurements. The second part addresses interfacial electronic structures of several model heterostructures in current condensed matter research using XSTM. Topics to be discussed include high-κ‘s/III-V’s semiconductors, polymer heterojunctions, and complex oxide heterostructures, which are all material systems whose investigation using this technique is expected to benefit the research community. Finally, practical aspects and perspectives of using XSTM in interface science are presented.

Correlating Intravital Multi-Photon Microscopy to 3D Electron Microscopy of Invading Tumor Cells Using Anatomical Reference Points

PubMed Central

Karreman, Matthia A.; Mercier, Luc; Schieber, Nicole L.; Shibue, Tsukasa; Schwab, Yannick; Goetz, Jacky G.

2014-01-01

Correlative microscopy combines the advantages of both light and electron microscopy to enable imaging of rare and transient events at high resolution. Performing correlative microscopy in complex and bulky samples such as an entire living organism is a time-consuming and error-prone task. Here, we investigate correlative methods that rely on the use of artificial and endogenous structural features of the sample as reference points for correlating intravital fluorescence microscopy and electron microscopy. To investigate tumor cell behavior in vivo with ultrastructural accuracy, a reliable approach is needed to retrieve single tumor cells imaged deep within the tissue. For this purpose, fluorescently labeled tumor cells were subcutaneously injected into a mouse ear and imaged using two-photon-excitation microscopy. Using near-infrared branding, the position of the imaged area within the sample was labeled at the skin level, allowing for its precise recollection. Following sample preparation for electron microscopy, concerted usage of the artificial branding and anatomical landmarks enables targeting and approaching the cells of interest while serial sectioning through the specimen. We describe here three procedures showing how three-dimensional (3D) mapping of structural features in the tissue can be exploited to accurately correlate between the two imaging modalities, without having to rely on the use of artificially introduced markers of the region of interest. The methods employed here facilitate the link between intravital and nanoscale imaging of invasive tumor cells, enabling correlating function to structure in the study of tumor invasion and metastasis. PMID:25479106

Applications of emerging transmission electron microscopy technology in PCD research and diagnosis.

PubMed

Shoemark, Amelia

2017-01-01

Primary Ciliary Dyskinesia (PCD) is a heterogeneous genetic condition characterized by dysfunction of motile cilia. Patients suffer from chronic infection and inflammation of the upper and lower respiratory tract. Diagnosis of PCD is confirmed by identification of a hallmark defect of ciliary ultrastructure or by identification of biallelic pathogenic mutations in a known PCD gene. Since the first description of PCD in 1976, assessment of ciliary ultrastructure by transmission electron microscopy (TEM) has been central to diagnosis and research. Electron tomography is a technique whereby a series of transmission electron micrographs are collected at different angles and reconstructed into a single 3D model of a specimen. Electron tomography provides improved spatial information and resolution compared to a single micrograph. Research by electron tomography has revealed new insight into ciliary ultrastructure and consequently ciliary function at a molecular and cellular level. Gene discovery studies in PCD have utilized electron tomography to define the structural consequences of variants in cilia genes. Modern transmission electron microscopes capable of electron tomography are increasingly being installed in clinical laboratories. This presents the possibility for the use of tomography technique in a diagnostic setting. This review describes the electron tomography technique, the contribution tomography has made to the understanding of basic cilia structure and function and finally the potential of the technique for use in PCD diagnosis.

Simulated electron beam trajectories toward a field ion microscopy specimen

NASA Astrophysics Data System (ADS)

Larson, D. J.; Camus, P. P.; Kelly, T. F.

1993-04-01

This article explores the conditions under which a directed electron beam originating nearly normal to the specimen axis can be made to impact the near-apex region of a field ion microscopy specimen in a high electric field. Electron trajectories were calculated using a modified Runge-Kutta numerical method. The results indicate that an electron beam can be directed to a specimen under typical field ion microscopy conditions using two methods: by varying initial beam tilt (less than 60 mrad) or by translating the initial beam position relative to the specimen apex (less than 5 mm). The net focusing effect of the high electric field on the electron beam can be treated, to first order, as an astigmatism and may be correctable by a post-lens deflection system.

Breaking resolution limits in ultrafast electron diffraction and microscopy.

PubMed

Baum, Peter; Zewail, Ahmed H

2006-10-31

Ultrafast electron microscopy and diffraction are powerful techniques for the study of the time-resolved structures of molecules, materials, and biological systems. Central to these approaches is the use of ultrafast coherent electron packets. The electron pulses typically have an energy of 30 keV for diffraction and 100-200 keV for microscopy, corresponding to speeds of 33-70% of the speed of light. Although the spatial resolution can reach the atomic scale, the temporal resolution is limited by the pulse width and by the difference in group velocities of electrons and the light used to initiate the dynamical change. In this contribution, we introduce the concept of tilted optical pulses into diffraction and imaging techniques and demonstrate the methodology experimentally. These advances allow us to reach limits of time resolution down to regimes of a few femtoseconds and, possibly, attoseconds. With tilted pulses, every part of the sample is excited at precisely the same time as when the electrons arrive at the specimen. Here, this approach is demonstrated for the most unfavorable case of ultrafast crystallography. We also present a method for measuring the duration of electron packets by autocorrelating electron pulses in free space and without streaking, and we discuss the potential of tilting the electron pulses themselves for applications in domains involving nuclear and electron motions.

Breaking resolution limits in ultrafast electron diffraction and microscopy

PubMed Central

Baum, Peter; Zewail, Ahmed H.

2006-01-01

Ultrafast electron microscopy and diffraction are powerful techniques for the study of the time-resolved structures of molecules, materials, and biological systems. Central to these approaches is the use of ultrafast coherent electron packets. The electron pulses typically have an energy of 30 keV for diffraction and 100–200 keV for microscopy, corresponding to speeds of 33–70% of the speed of light. Although the spatial resolution can reach the atomic scale, the temporal resolution is limited by the pulse width and by the difference in group velocities of electrons and the light used to initiate the dynamical change. In this contribution, we introduce the concept of tilted optical pulses into diffraction and imaging techniques and demonstrate the methodology experimentally. These advances allow us to reach limits of time resolution down to regimes of a few femtoseconds and, possibly, attoseconds. With tilted pulses, every part of the sample is excited at precisely the same time as when the electrons arrive at the specimen. Here, this approach is demonstrated for the most unfavorable case of ultrafast crystallography. We also present a method for measuring the duration of electron packets by autocorrelating electron pulses in free space and without streaking, and we discuss the potential of tilting the electron pulses themselves for applications in domains involving nuclear and electron motions. PMID:17056711

Low-voltage electron microscopy of polymer and organic molecular thin films.

PubMed

Drummy, Lawrence F; Yang, Junyan; Martin, David C

2004-06-01

We have demonstrated the capabilities of a novel low-voltage electron microscope (LVEM) for imaging polymer and organic molecular thin films. The LVEM can operate in transmission electron microscopy, scanning transmission electron microscopy, scanning electron microscopy, and electron diffraction modes. The microscope operates at a nominal accelerating voltage of 5 kV and fits on a tabletop. A detailed discussion of the electron-sample interaction processes is presented, and the mean free path for total electron scattering was calculated to be 15 nm for organic samples at 5 kV. The total end point dose for the destruction of crystallinity at 5 kV was estimated at 5 x 10(-4) and 3.5 x 10(-2) C/cm2 for polyethylene and pentacene, respectively. These values are significantly lower than those measured at voltages greater than 100 kV. A defocus series of colloidal gold particles allowed us to estimate the experimental contrast transfer function of the microscope. Images taken of several organic materials have shown high contrast for low atomic number elements and a resolution of 2.5 nm. The materials studied here include thin films of the organic semiconductor pentacene, triblock copolymer films, single-molecule dendrimers, electrospun polymer fibers and gold nanoparticles. Copyright 2004 Elsevier B.V.

Nanofabrication by advanced electron microscopy using intense and focused beam∗.

PubMed

Furuya, Kazuo

2008-01-01

The nanogrowth and nanofabrication of solid substances using an intense and focused electron beam are reviewed in terms of the application of scanning and transmission electron microscopy (SEM, TEM and STEM) to control the size, position and structure of nanomaterials. The first example discussed is the growth of freestanding nanotrees on insulator substrates by TEM. The growth process of the nanotrees was observed in situ and analyzed by high-resolution TEM (HRTEM) and was mainly controlled by the intensity of the electron beam. The second example is position- and size-controlled nanofabrication by STEM using a focused electron beam. The diameters of the nanostructures grown ranged from 4 to 20 nm depending on the size of the electron beam. Magnetic nanostructures were also obtained using an iron-containing precursor gas, Fe(CO) 5 . The freestanding iron nanoantennas were examined by electron holography. The magnetic field was observed to leak from the nanostructure body which appeared to act as a 'nanomagnet'. The third example described is the effect of a vacuum on the size and growth process of fabricated nanodots containing W in an ultrahigh-vacuum field-emission TEM (UHV-FE-TEM). The size of the dots can be controlled by changing the dose of electrons and the partial pressure of the precursor. The smallest particle size obtained was about 1.5 nm in diameter, which is the smallest size reported using this method. Finally, the importance of a smaller probe and a higher electron-beam current with atomic resolution is emphasized and an attempt to develop an ultrahigh-vacuum spherical aberration corrected STEM (Cs-corrected STEM) at NIMS is reported.

Nanofabrication by advanced electron microscopy using intense and focused beam∗

PubMed Central

Furuya, Kazuo

2008-01-01

The nanogrowth and nanofabrication of solid substances using an intense and focused electron beam are reviewed in terms of the application of scanning and transmission electron microscopy (SEM, TEM and STEM) to control the size, position and structure of nanomaterials. The first example discussed is the growth of freestanding nanotrees on insulator substrates by TEM. The growth process of the nanotrees was observed in situ and analyzed by high-resolution TEM (HRTEM) and was mainly controlled by the intensity of the electron beam. The second example is position- and size-controlled nanofabrication by STEM using a focused electron beam. The diameters of the nanostructures grown ranged from 4 to 20 nm depending on the size of the electron beam. Magnetic nanostructures were also obtained using an iron-containing precursor gas, Fe(CO)5. The freestanding iron nanoantennas were examined by electron holography. The magnetic field was observed to leak from the nanostructure body which appeared to act as a ‘nanomagnet’. The third example described is the effect of a vacuum on the size and growth process of fabricated nanodots containing W in an ultrahigh-vacuum field-emission TEM (UHV-FE-TEM). The size of the dots can be controlled by changing the dose of electrons and the partial pressure of the precursor. The smallest particle size obtained was about 1.5 nm in diameter, which is the smallest size reported using this method. Finally, the importance of a smaller probe and a higher electron-beam current with atomic resolution is emphasized and an attempt to develop an ultrahigh-vacuum spherical aberration corrected STEM (Cs-corrected STEM) at NIMS is reported. PMID:27877936

Tackling the Challenges of Dynamic Experiments Using Liquid-Cell Transmission Electron Microscopy.

PubMed

Parent, Lucas R; Bakalis, Evangelos; Proetto, Maria; Li, Yiwen; Park, Chiwoo; Zerbetto, Francesco; Gianneschi, Nathan C

2018-01-16

Revolutions in science and engineering frequently result from the development, and wide adoption, of a new, powerful characterization or imaging technique. Beginning with the first glass lenses and telescopes in astronomy, to the development of visual-light microscopy, staining techniques, confocal microscopy, and fluorescence super-resolution microscopy in biology, and most recently aberration-corrected, cryogenic, and ultrafast (4D) electron microscopy, X-ray microscopy, and scanning probe microscopy in nanoscience. Through these developments, our perception and understanding of the physical nature of matter at length-scales beyond ordinary perception have been fundamentally transformed. Despite this progression in microscopy, techniques for observing nanoscale chemical processes and solvated/hydrated systems are limited, as the necessary spatial and temporal resolution presents significant technical challenges. However, the standard reliance on indirect or bulk phase characterization of nanoscale samples in liquids is undergoing a shift in recent times with the realization ( Williamson et al. Nat. Mater . 2003 , 2 , 532 - 536 ) of liquid-cell (scanning) transmission electron microscopy, LC(S)TEM, where picoliters of solution are hermetically sealed between electron-transparent "windows," which can be directly imaged or videoed at the nanoscale using conventional transmission electron microscopes. This Account seeks to open a discussion on the topic of standardizing strategies for conducting imaging experiments with a view to characterizing dynamics and motion of nanoscale materials. This is a challenge that could be described by critics and proponents alike, as analogous to doing chemistry in a lightning storm; where the nature of the solution, the nanomaterial, and the dynamic behaviors are all potentially subject to artifactual influence by the very act of our observation.

New hardware and workflows for semi-automated correlative cryo-fluorescence and cryo-electron microscopy/tomography.

PubMed

Schorb, Martin; Gaechter, Leander; Avinoam, Ori; Sieckmann, Frank; Clarke, Mairi; Bebeacua, Cecilia; Bykov, Yury S; Sonnen, Andreas F-P; Lihl, Reinhard; Briggs, John A G

2017-02-01

Correlative light and electron microscopy allows features of interest defined by fluorescence signals to be located in an electron micrograph of the same sample. Rare dynamic events or specific objects can be identified, targeted and imaged by electron microscopy or tomography. To combine it with structural studies using cryo-electron microscopy or tomography, fluorescence microscopy must be performed while maintaining the specimen vitrified at liquid-nitrogen temperatures and in a dry environment during imaging and transfer. Here we present instrumentation, software and an experimental workflow that improves the ease of use, throughput and performance of correlated cryo-fluorescence and cryo-electron microscopy. The new cryo-stage incorporates a specially modified high-numerical aperture objective lens and provides a stable and clean imaging environment. It is combined with a transfer shuttle for contamination-free loading of the specimen. Optimized microscope control software allows automated acquisition of the entire specimen area by cryo-fluorescence microscopy. The software also facilitates direct transfer of the fluorescence image and associated coordinates to the cryo-electron microscope for subsequent fluorescence-guided automated imaging. Here we describe these technological developments and present a detailed workflow, which we applied for automated cryo-electron microscopy and tomography of various specimens. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Transmission electron microscopy of the preclinical phase of experimental phytophotodermatitis.

PubMed

Almeida, Hiram Larangeira de; Sotto, Miriam Nakagami; Castro, Luis Antonio Suita de; Rocha, Nara Moreira

2008-06-01

To examine the epidermis in induced phytophotodermatitis using transmission electron microscopy in order to detect histologic changes even before lesions are visible by light microscopy. In the first six hours after the experimental induction of phytophotodermatitis, no changes are detectable by light microscopy. Only after 24 hours can keratinocyte necrosis and epidermal vacuolization be detected histologically, and blisters form by 48 hours. The dorsum of four adult rats (Rattus norvegicus) was manually epilated. After painting the right half of the rat with the peel juice of Tahiti lemon, they were exposed to sunlight for eight minutes under general anesthesia. The left side was used as the control and exposed to sunlight only. Biopsies were performed immediately after photoinduction and one and two hours later, and the tissue was analyzed by transmission electron microscopy. No histological changes were seen on the control side. Immediately after induction, vacuolization in keratinocytes was observed. After one hour, desmosomal changes were also observed in addition to vacuolization. Keratin filaments were not attached to the desmosomal plaque. Free desmosomes and membrane ruptures were also seen. At two hours after induction, similar changes were found, and granular degeneration of keratin was also observed. The interaction of sunlight and psoralens generates a photoproduct that damages keratinocyte proteins, leading to keratinocyte necrosis and blister formation. Transmission electron microscopy can detect vacuolization, lesions of the membrane, and desmosomes in the first two hours after experimental induction of phytophotodermatitis.

Epoxy Resins in Electron Microscopy

PubMed Central

Finck, Henry

1960-01-01

A method of embedding biological specimens in araldite 502 (Ciba) has been developed for materials available in the United States. Araldite-embedded tissues are suitable for electron microscopy, but the cutting qualities of the resin necessitates more than routine attention during microtomy. The rather high viscosity of araldite 502 also seems to be an unnecessary handicap. The less viscous epoxy epon 812 (Shell) produces specimens with improved cutting qualities, and has several features—low shrinkage and absence of specimen damage during cure, minimal compression of sections, relative absence of electron beam-induced section damage, etc.—which recommends it as a routine embedding material. The hardness of the cured resin can be easily adjusted by several methods to suit the materials embedded in it. Several problems and advantages of working with sections of epoxy resins are also discussed. PMID:13822825

ELECTRON MICROSCOPY OF HELA CELLS INFECTED WITH ADENOVIRUSES

PubMed Central

Harford, Carl G.; Hamlin, Alice; Parker, Esther; van Ravenswaay, Theodore

1956-01-01

HeLa cells were infected with adenoviruses (types 1–4) and sectioned for electron microscopy after intervals of 20 to 48 hours. Clusters of virus-like particles were found within the nuclei of infected cultures but not in those of uninfected controls. The particles were often arranged in rows as if in crystalline formation. Maximal diameter of particles was approximately 65 mµ, and internal bodies were demonstrated. Lesions of infected cells included target-like structures of the nuclear membrane, large nuclear vacuoles (type 2), and increased numbers of large irregular electron-dense granules in the cytoplasm 48 hours after infection. Examination of infected cultures by light microscopy, using the Feulgen reaction, showed intranuclear inclusion bodies and a cytopathogenic effect consisting of clumping of cells without pyknosis of nuclei. A lipide stain showed numerous cytoplasmic granules that were not identical with the large, irregular, electron-dense granules of the cytoplasm. Practically all the cells showed the viral cytopathogenic effect, but only a minority of cells were found to contain virus-like particles or intranuclear inclusion bodies. PMID:13357696

Electron Microscopy of Living Cells During in Situ Fluorescence Microscopy

PubMed Central

Liv, Nalan; van Oosten Slingeland, Daan S. B.; Baudoin, Jean-Pierre; Kruit, Pieter; Piston, David W.; Hoogenboom, Jacob P.

2016-01-01

We present an approach toward dynamic nanoimaging: live fluorescence of cells encapsulated in a bionanoreactor is complemented with in situ scanning electron microscopy (SEM) on an integrated microscope. This allows us to take SEM snapshots on-demand, that is, at a specific location in time, at a desired region of interest, guided by the dynamic fluorescence imaging. We show that this approach enables direct visualization, with EM resolution, of the distribution of bioconjugated quantum dots on cellular extensions during uptake and internalization. PMID:26580231

Combined time-lapse cinematography and immuno-electron microscopy.

PubMed

Balfour, B M; Goscicka, T; MacKenzie, J L; Gautam, A; Tate, M; Clark, J

1990-04-01

A method was developed to record interactions between mobile non-adherent immunocytes by time-lapse cinematography and then to study the same cells by immuno-electron microscopy, using monoclonal antibodies against surface components. For this purpose a modified stage was designed to fit an inverted microscope. The attachment included a device to cool the culture chamber with N2 gas, a micro-injector for monoclonal antibody and immuno-gold treatment, and two pairs of washing needles to change the medium without disturbance. The technique was first employed to study the formation of aggregates around the antigen-presenting cells in cultures containing cells from hyper-immunized animals. Recently peripheral blood cells from normal subjects and patients with immune deficiency syndromes were stimulated with pokeweed mitogen, cluster formation was recorded, and the cells were processed for immuno-electron microscopy.

Ultrafast electron microscopy integrated with a direct electron detection camera.

PubMed

Lee, Young Min; Kim, Young Jae; Kim, Ye-Jin; Kwon, Oh-Hoon

2017-07-01

In the past decade, we have witnessed the rapid growth of the field of ultrafast electron microscopy (UEM), which provides intuitive means to watch atomic and molecular motions of matter. Yet, because of the limited current of the pulsed electron beam resulting from space-charge effects, observations have been mainly made to periodic motions of the crystalline structure of hundreds of nanometers or higher by stroboscopic imaging at high repetition rates. Here, we develop an advanced UEM with robust capabilities for circumventing the present limitations by integrating a direct electron detection camera for the first time which allows for imaging at low repetition rates. This approach is expected to promote UEM to a more powerful platform to visualize molecular and collective motions and dissect fundamental physical, chemical, and materials phenomena in space and time.

ATOMIC RESOLUTION CRYO ELECTRON MICROSCOPY OF MACROMOLECULAR COMPLEXES

PubMed Central

ZHOU, Z. HONG

2013-01-01

Single-particle cryo electron microscopy (cryoEM) is a technique for determining three-dimensional (3D) structures from projection images of molecular complexes preserved in their “native,” noncrystalline state. Recently, atomic or near-atomic resolution structures of several viruses and protein assemblies have been determined by single-particle cryoEM, allowing ab initio atomic model building by following the amino acid side chains or nucleic acid bases identifiable in their cryoEM density maps. In particular, these cryoEM structures have revealed extended arms contributing to molecular interactions that are otherwise not resolved by the conventional structural method of X-ray crystallography at similar resolutions. High-resolution cryoEM requires careful consideration of a number of factors, including proper sample preparation to ensure structural homogeneity, optimal configuration of electron imaging conditions to record high-resolution cryoEM images, accurate determination of image parameters to correct image distortions, efficient refinement and computation to reconstruct a 3D density map, and finally appropriate choice of modeling tools to construct atomic models for functional interpretation. This progress illustrates the power of cryoEM and ushers it into the arsenal of structural biology, alongside conventional techniques of X-ray crystallography and NMR, as a major tool (and sometimes the preferred one) for the studies of molecular interactions in supramolecular assemblies or machines. PMID:21501817

Evaluation of a hybrid pixel detector for electron microscopy.

PubMed

Faruqi, A R; Cattermole, D M; Henderson, R; Mikulec, B; Raeburn, C

2003-04-01

We describe the application of a silicon hybrid pixel detector, containing 64 by 64 pixels, each 170 microm(2), in electron microscopy. The device offers improved resolution compared to CCDs along with faster and noiseless readout. Evaluation of the detector, carried out on a 120 kV electron microscope, demonstrates the potential of the device.

Atomic-resolution transmission electron microscopy of electron beam–sensitive crystalline materials

NASA Astrophysics Data System (ADS)

Zhang, Daliang; Zhu, Yihan; Liu, Lingmei; Ying, Xiangrong; Hsiung, Chia-En; Sougrat, Rachid; Li, Kun; Han, Yu

2018-02-01

High-resolution imaging of electron beam–sensitive materials is one of the most difficult applications of transmission electron microscopy (TEM). The challenges are manifold, including the acquisition of images with extremely low beam doses, the time-constrained search for crystal zone axes, the precise image alignment, and the accurate determination of the defocus value. We develop a suite of methods to fulfill these requirements and acquire atomic-resolution TEM images of several metal organic frameworks that are generally recognized as highly sensitive to electron beams. The high image resolution allows us to identify individual metal atomic columns, various types of surface termination, and benzene rings in the organic linkers. We also apply our methods to other electron beam–sensitive materials, including the organic-inorganic hybrid perovskite CH3NH3PbBr3.

Investigation of Nematode Diversity using Scanning Electron Microscopy and Fluorescent Microscopy

NASA Astrophysics Data System (ADS)

Seacor, Taylor; Howell, Carina

2013-03-01

Nematode worms account for the vast majority of the animals in the biosphere. They are colossally important to global public health as parasites, and to agriculture both as pests and as beneficial inhabitants of healthy soil. Amphid neurons are the anterior chemosensory neurons in nematodes, mediating critical behaviors including chemotaxis and mating. We are examining the cellular morphology and external anatomy of amphid neurons, using fluorescence microscopy and scanning electron microscopy, respectively, of a wide range of soil nematodes isolated in the wild. We use both classical systematics (e.g. diagnostic keys) and molecular markers (e.g. ribosomal RNA) to classify these wild isolates. Our ultimate aim is to build a detailed anatomical database in order to dissect genetic pathways of neuronal development and function across phylogeny and ecology. Research supported by NSF grants 092304, 0806660, 1058829 and Lock Haven University FPDC grants

Nanowire growth kinetics in aberration corrected environmental transmission electron microscopy

DOE PAGES

Chou, Yi -Chia; Panciera, Federico; Reuter, Mark C.; ...

2016-03-15

Here, we visualize atomic level dynamics during Si nanowire growth using aberration corrected environmental transmission electron microscopy, and compare with lower pressure results from ultra-high vacuum microscopy. We discuss the importance of higher pressure observations for understanding growth mechanisms and describe protocols to minimize effects of the higher pressure background gas.

In Situ Electron Microscopy of Lactomicroselenium Particles in Probiotic Bacteria.

PubMed

Nagy, Gabor; Pinczes, Gyula; Pinter, Gabor; Pocsi, Istvan; Prokisch, Jozsef; Banfalvi, Gaspar

2016-06-30

Electron microscopy was used to test whether or not (a) in statu nascendi synthesized, and in situ measured, nanoparticle size does not differ significantly from the size of nanoparticles after their purification; and (b) the generation of selenium is detrimental to the bacterial strains that produce them. Elemental nano-sized selenium produced by probiotic latic acid bacteria was used as a lactomicroselenium (lactomicroSel) inhibitor of cell growth in the presence of lactomicroSel, and was followed by time-lapse microscopy. The size of lactomicroSel produced by probiotic bacteria was measured in situ and after isolation and purification. For these measurements the TESLA BS 540 transmission electron microscope was converted from analog (aTEM) to digital processing (dTEM), and further to remote-access internet electron microscopy (iTEM). Lactobacillus acidophilus produced fewer, but larger, lactomicroSel nanoparticles (200-350 nm) than Lactobacillus casei (L. casei), which generated many, smaller lactomicroSel particles (85-200 nm) and grains as a cloudy, less electrodense material. Streptococcus thermophilus cells generated selenoparticles (60-280 nm) in a suicidic manner. The size determined in situ in lactic acid bacteria was significantly lower than those measured by scanning electron microscopy after the isolation of lactomicroSel particles obtained from lactobacilli (100-500 nm), but higher relative to those isolated from Streptococcus thermopilus (50-100 nm). These differences indicate that smaller lactomicroSel particles could be more toxic to the producing bacteria themselves and discrepancies in size could have implications with respect to the applications of selenium nanoparticles as prebiotics.

Electron microscopy study of antioxidant interaction with bacterial cells

NASA Astrophysics Data System (ADS)

Plotnikov, Oleg P.; Novikova, Olga V.; Konnov, Nikolai P.; Korsukov, Vladimir N.; Gunkin, Ivan F.; Volkov, Uryi P.

2000-10-01

To maintain native microorganisms genotype and phenotype features a lyophylization technique is widely used. However in this case cells are affected by influences of vacuum and low temperature that cause a part of the cells population to be destruction. Another factor reduced microorganisms vitality is formation of reactive oxygen forms that damage certain biological targets (such as DNA, membranes etc.) Recently to raise microorganism's resistance against adverse condition natural and synthetic antioxidants are used. Antioxidant- are antagonists of free radicals. Introduction of antioxidants in protective medium for lyophylization increase bacteria storage life about 2,0-4,8 fold in comparison with reference samples. In the article the main results of our investigation of antioxidants interaction with microorganism cells is described. As bacteria cells we use vaccine strain yersinia pestis EV, that were grown for 48 h at 28 degree(s)C on the Hottinger agar (pH 7,2). Antioxidants are inserted on the agar surface in specimen under test. To investigate a localization of antioxidants for electron microscopy investigation, thallium organic antioxidants were used. The thallium organic compounds have an antioxidant features if thallium is in low concentration (about 1(mu) g/ml). The localization of the thallium organic antioxidants on bacteria Y. pestis EV is visible in electron microscopy images, thallium being heavy metal with high electron density. The negatively stained bacteria and bacteria thin sections with thallium organic compounds were investigated by means of transmission electron microscopy. The localization of the thallium organic compounds is clearly visible in electron micrographs as small dark spots with size about 10-80nm. Probably mechanisms of interaction of antioxidants with bacteria cells are discussed.

Three dimensional electron microscopy and in silico tools for macromolecular structure determination

PubMed Central

Borkotoky, Subhomoi; Meena, Chetan Kumar; Khan, Mohammad Wahab; Murali, Ayaluru

2013-01-01

Recently, structural biology witnessed a major tool - electron microscopy - in solving the structures of macromolecules in addition to the conventional techniques, X-ray crystallography and nuclear magnetic resonance (NMR). Three dimensional transmission electron microscopy (3DTEM) is one of the most sophisticated techniques for structure determination of molecular machines. Known to give the 3-dimensional structures in its native form with literally no upper limit on size of the macromolecule, this tool does not need the crystallization of the protein. Combining the 3DTEM data with in silico tools, one can have better refined structure of a desired complex. In this review we are discussing about the recent advancements in three dimensional electron microscopy and tools associated with it. PMID:27092033

Probing Structural and Electronic Dynamics with Ultrafast Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Plemmons, DA; Suri, PK; Flannigan, DJ

In this Perspective, we provide an overview,of the field of ultrafast electron microscopy (UEM). We begin by briefly discussing the emergence of methods for probing ultrafast structural dynamics and the information that can be obtained. Distinctions are drawn between the two main types a probes for femtosecond (fs) dynamics fast electrons and X-ray photons and emphasis is placed on hour the nature of charged particles is exploited in ultrafast electron-based' experiments:. Following this, we describe the versatility enabled by the ease with which electron trajectories and velocities can be manipulated with transmission electron microscopy (TEM): hardware configurations, and we emphasizemore » how this is translated to the ability to measure scattering intensities in real, reciprocal, and energy space from presurveyed and selected rianoscale volumes. Owing to decades of ongoing research and development into TEM instrumentation combined with advances in specimen holder technology, comprehensive experiments can be conducted on a wide range of materials in various phases via in situ methods. Next, we describe the basic operating concepts, of UEM, and we emphasize that its development has led to extension of several of the formidable capabilities of TEM into the fs domain, dins increasing the accessible temporal parameter spade by several orders of magnitude. We then divide UEM studies into those conducted in real (imaging), reciprocal (diffraction), and energy (spectroscopy) spate. We begin each of these sections by providing a brief description of the basic operating principles and the types of information that can be gathered followed by descriptions of how these approaches are applied in UM, the type of specimen parameter space that can be probed, and an example of the types of dynamics that can be resolved. We conclude with an Outlook section, wherein we share our perspective on some future directions of the field pertaining to continued instrument development

The spatial coherence function in scanning transmission electron microscopy and spectroscopy.

PubMed

Nguyen, D T; Findlay, S D; Etheridge, J

2014-11-01

We investigate the implications of the form of the spatial coherence function, also referred to as the effective source distribution, for quantitative analysis in scanning transmission electron microscopy, and in particular for interpreting the spatial origin of imaging and spectroscopy signals. These questions are explored using three different source distribution models applied to a GaAs crystal case study. The shape of the effective source distribution was found to have a strong influence not only on the scanning transmission electron microscopy (STEM) image contrast, but also on the distribution of the scattered electron wavefield and hence on the spatial origin of the detected electron intensities. The implications this has for measuring structure, composition and bonding at atomic resolution via annular dark field, X-ray and electron energy loss STEM imaging are discussed. Copyright © 2014 Elsevier B.V. All rights reserved.

High-resolution low-dose scanning transmission electron microscopy.

PubMed

Buban, James P; Ramasse, Quentin; Gipson, Bryant; Browning, Nigel D; Stahlberg, Henning

2010-01-01

During the past two decades instrumentation in scanning transmission electron microscopy (STEM) has pushed toward higher intensity electron probes to increase the signal-to-noise ratio of recorded images. While this is suitable for robust specimens, biological specimens require a much reduced electron dose for high-resolution imaging. We describe here protocols for low-dose STEM image recording with a conventional field-emission gun STEM, while maintaining the high-resolution capability of the instrument. Our findings show that a combination of reduced pixel dwell time and reduced gun current can achieve radiation doses comparable to low-dose TEM.

Nanomusical systems visualized and controlled in 4D electron microscopy.

PubMed

Baskin, J Spencer; Park, Hyun Soon; Zewail, Ahmed H

2011-05-11

Nanomusical systems, nanoharp and nanopiano, fabricated as arrays of cantilevers by focused ion beam milling of a layered Ni/Ti/Si(3)N(4) thin film, have been investigated in 4D electron microscopy. With the imaging and selective femtosecond and nanosecond control combinations, full characterization of the amplitude and phase of the resonant response of a particular cantilever relative to the optical pulse train was possible. Using a high repetition rate, low energy optical pulse train for selective, resonant excitation, coupled with pulsed and steady-state electron imaging for visualization in space and time, both the amplitude on the nanoscale and resonance of motion on the megahertz scale were resolved for these systems. Tilting of the specimen allowed in-plane and out-of-plane cantilever bending and cantilever torsional motions to be identified in stroboscopic measurements of impulsively induced free vibration. Finally, the transient, as opposed to steady state, thermostat effect was observed for the layered nanocantilevers, with a sufficiently sensitive response to demonstrate suitability for in situ use in thin-film temperature measurements requiring resolutions of <10 K and 10 μm on time scales here mechanically limited to microseconds and potentially at shorter times.

A Dose-Rate Effect in Single-Particle Electron Microscopy

PubMed Central

Chen, James Z.; Sachse, Carsten; Xu, Chen; Mielke, Thorsten; Spahn, Christian M. T.; Grigorieff, Nikolaus

2008-01-01

A low beam-intensity, low electron-dose imaging method has been developed for single-particle electron cryo-microscopy (cryo-EM). Experiments indicate that the new technique can reduce beam-induced specimen movement and secondary radiolytic effects, such as “bubbling”. The improvement in image quality, especially for multiple-exposure data collection, will help single-particle cryo-EM to reach higher resolution. PMID:17977018

A Mobile Nanoscience and Electron Microscopy Outreach Program

NASA Astrophysics Data System (ADS)

Coffey, Tonya; Kelley, Kyle

2013-03-01

We have established a mobile nanoscience laboratory outreach program in Western NC that puts scanning electron microscopy (SEM) directly in the hands of K-12 students and the general public. There has been a recent push to develop new active learning materials to educate students at all levels about nanoscience and nanotechnology. Previous projects, such as Bugscope, nanoManipulator, or SPM Live! allowed remote access to advanced microscopies. However, placing SEM directly in schools has not often been possible because the cost and steep learning curve of these technologies were prohibitive, making this project quite novel. We have developed new learning modules for a microscopy outreach experience with a tabletop SEM (Hitachi TM3000). We present here an overview of our outreach and results of the assessment of our program to date.

Electron microscopy approach for the visualization of the epithelial and endothelial glycocalyx.

PubMed

Chevalier, L; Selim, J; Genty, D; Baste, J M; Piton, N; Boukhalfa, I; Hamzaoui, M; Pareige, P; Richard, V

2017-06-01

This study presents a methodological approach for the visualization of the glycocalyx by electron microscopy. The glycocalyx is a three dimensional network mainly composed of glycolipids, glycoproteins and proteoglycans associated with the plasma membrane. Since less than a decade, the epithelial and endothelial glycocalyx proved to play an important role in physiology and pathology, increasing its research interest especially in vascular functions. Therefore, visualization of the glycocalyx requires reliable techniques and its preservation remains challenging due to its fragile and dynamic organization, which is highly sensitive to the different process steps for electron microscopy sampling. In this study, chemical fixation was performed by perfusion as a good alternative to conventional fixation. Additional lanthanum nitrate in the fixative enhances staining of the glycocalyx in transmission electron microscopy bright field and improves its visualization by detecting the elastic scattered electrons, thus providing a chemical contrast. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Navigating 3D electron microscopy maps with EM-SURFER.

PubMed

Esquivel-Rodríguez, Juan; Xiong, Yi; Han, Xusi; Guang, Shuomeng; Christoffer, Charles; Kihara, Daisuke

2015-05-30

The Electron Microscopy DataBank (EMDB) is growing rapidly, accumulating biological structural data obtained mainly by electron microscopy and tomography, which are emerging techniques for determining large biomolecular complex and subcellular structures. Together with the Protein Data Bank (PDB), EMDB is becoming a fundamental resource of the tertiary structures of biological macromolecules. To take full advantage of this indispensable resource, the ability to search the database by structural similarity is essential. However, unlike high-resolution structures stored in PDB, methods for comparing low-resolution electron microscopy (EM) density maps in EMDB are not well established. We developed a computational method for efficiently searching low-resolution EM maps. The method uses a compact fingerprint representation of EM maps based on the 3D Zernike descriptor, which is derived from a mathematical series expansion for EM maps that are considered as 3D functions. The method is implemented in a web server named EM-SURFER, which allows users to search against the entire EMDB in real-time. EM-SURFER compares the global shapes of EM maps. Examples of search results from different types of query structures are discussed. We developed EM-SURFER, which retrieves structurally relevant matches for query EM maps from EMDB within seconds. The unique capability of EM-SURFER to detect 3D shape similarity of low-resolution EM maps should prove invaluable in structural biology.

On the state of crystallography at the dawn of the electron microscopy revolution.

PubMed

Higgins, Matthew K; Lea, Susan M

2017-10-01

While protein crystallography has, for many years, been the most used method for structural analysis of macromolecular complexes, remarkable recent advances in high-resolution electron cryo-microscopy led to suggestions that 'the revolution will not be crystallised'. Here we highlight the current success rate, speed and ease of modern crystallographic structure determination and some recent triumphs of both 'classical' crystallography and the use of X-ray free electron lasers. We also outline fundamental differences between structure determination using X-ray crystallography and electron microscopy. We suggest that crystallography will continue to co-exist with electron microscopy as part of an integrated array of methods, allowing structural biologists to focus on fundamental biological questions rather than being constrained by the methods available. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Electron Microscopy of Ultrathin Sections of Sporosarcina ureae

PubMed Central

Mazanec, K.; Kocur, M.; Martinec, T.

1965-01-01

Mazanec, K. (J. E. Purkyně University, Brno, Czechoslovakia), M. Kocur, and T. Martinec. Electron microscopy of ultrathin sections of Sporosarcina ureae. J. Bacteriol. 90:808–816. 1965.—Ultrathin sections of Sporosarcina ureae cells were studied by means of electron microscopy. The cell wall consists of several layers and is 340 A thick. The cytoplasm is of globular structure and includes ribosomelike structures, occasional mesosomes, and inclusions not precisely identifiable. The nuclear area has various shapes and is formed by filaments 10 to 20 A thick which proceed in various directions. Cell division occurs similarly to that of sarcinate. Both synchronic and asynchronic cell division was observed. The spores of S. ureae consist of an outer coat having several layers, a cortex, a spore wall, and cytoplasm. The results of the present investigation substantiate our previous suggestion that S. ureae should be transferred from the family Micrococcaceae to the family Bacillaceae. Images PMID:16562085

A national facility for biological cryo-electron microscopy

PubMed Central

Saibil, Helen R.; Grünewald, Kay; Stuart, David I.

2015-01-01

Three-dimensional electron microscopy is an enormously powerful tool for structural biologists. It is now able to provide an understanding of the molecular machinery of cells, disease processes and the actions of pathogenic organisms from atomic detail through to the cellular context. However, cutting-edge research in this field requires very substantial resources for equipment, infrastructure and expertise. Here, a brief overview is provided of the plans for a UK national three-dimensional electron-microscopy facility for integrated structural biology to enable internationally leading research on the machinery of life. State-of-the-art equipment operated with expert support will be provided, optimized for both atomic-level single-particle analysis of purified macromolecules and complexes and for tomography of cell sections. The access to and organization of the facility will be modelled on the highly successful macromolecular crystallography (MX) synchrotron beamlines, and will be embedded at the Diamond Light Source, facilitating the development of user-friendly workflows providing near-real-time experimental feedback. PMID:25615867

Heat- and electron-beam-induced transport of gold particles into silicon oxide and silicon studied by in situ high-resolution transmission electron microscopy.

PubMed

Biskupek, Johannes; Kaiser, Ute; Falk, Fritz

2008-06-01

In this study, we describe the transport of gold (Au) nanoparticles from the surface into crystalline silicon (Si) covered by silicon oxide (SiO(2)) as revealed by in situ high-resolution transmission electron microscopy. Complete crystalline Au nanoparticles sink through the SiO(2) layer into the Si substrate when high-dose electron irradiation is applied and temperature is raised above 150 degrees C. Above temperatures of 250 degrees C, the Au nanoparticles finally dissolve into fragments accompanied by crystallization of the amorphized Si substrate around these fragments. The transport process is explained by a wetting process followed by Stokes motion. Modelling this process yields boundaries for the interface energies involved.

Analysis of electromagnetic forces and causality in electron microscopy.

PubMed

Reyes-Coronado, Alejandro; Ortíz-Solano, Carlos Gael; Zabala, Nerea; Rivacoba, Alberto; Esquivel-Sirvent, Raúl

2018-09-01

The non-physical effects on the transverse momentum transfer from fast electrons to gold nanoparticles associated to the use of non-causal dielectric functions are studied. A direct test of the causality based on the surface Kramers-Kronig relations is presented. This test is applied to the different dielectric function used to describe gold nanostructures in electron microscopy. Copyright © 2018. Published by Elsevier B.V.

Chemical mapping and quantification at the atomic scale by scanning transmission electron microscopy.

PubMed

Chu, Ming-Wen; Chen, Cheng Hsuan

2013-06-25

With innovative modern material-growth methods, a broad spectrum of fascinating materials with reduced dimensions-ranging from single-atom catalysts, nanoplasmonic and nanophotonic materials to two-dimensional heterostructural interfaces-is continually emerging and extending the new frontiers of materials research. A persistent central challenge in this grand scientific context has been the detailed characterization of the individual objects in these materials with the highest spatial resolution, a problem prompting the need for experimental techniques that integrate both microscopic and spectroscopic capabilities. To date, several representative microscopy-spectroscopy combinations have become available, such as scanning tunneling microscopy, tip-enhanced scanning optical microscopy, atom probe tomography, scanning transmission X-ray microscopy, and scanning transmission electron microscopy (STEM). Among these tools, STEM boasts unique chemical and electronic sensitivity at unparalleled resolution. In this Perspective, we elucidate the advances in STEM and chemical mapping applications at the atomic scale by energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy with a focus on the ultimate challenge of chemical quantification with atomic accuracy.

Attosecond electron pulses for 4D diffraction and microscopy

PubMed Central

Baum, Peter; Zewail, Ahmed H.

2007-01-01

In this contribution, we consider the advancement of ultrafast electron diffraction and microscopy to cover the attosecond time domain. The concept is centered on the compression of femtosecond electron packets to trains of 15-attosecond pulses by the use of the ponderomotive force in synthesized gratings of optical fields. Such attosecond electron pulses are significantly shorter than those achievable with extreme UV light sources near 25 nm (≈50 eV) and have the potential for applications in the visualization of ultrafast electron dynamics, especially of atomic structures, clusters of atoms, and some materials. PMID:18000040

The sea urchin egg jelly coat is a three-dimensional fibrous network as seen by intermediate voltage electron microscopy and deep etching analysis.

PubMed

Bonnell, B S; Larabell, C; Chandler, D E

1993-06-01

The egg jelly (EJ) coat which surrounds the unfertilized sea urchin egg undergoes extensive swelling upon contact with sea water, forming a three-dimensional network of interconnected fibers extending nearly 50 microns from the egg surface. Owing to its solubility, this coat has been difficult to visualize by light and electron microscopy. However, Lytechinus pictus EJ coats remain intact, if the fixation medium is maintained at pH 9. The addition of alcian blue during the final dehydration step of sample preparation stains the EJ for visualization of resin embedded eggs by both light and electron microscopy. Stereo pairs taken of thick sections prepared for intermediate voltage electron microscopy (IVEM) produce a three-dimensional image of the EJ network, consisting of interconnected fibers decorated along their length by globular jelly components. Using scanning electron microscopy (SEM), we have shown that before swelling, EJ exists in a tightly bound network of jelly fibers, 50-60 nm in diameter. In contrast, swollen EJ consists of a greatly extended network whose fibrous components measure 10 to 30 nm in diameter. High resolution stereo images of hydrated jelly produced by the quick-freeze/deep-etch/rotary-shadowing technique (QF/DE/RS) show nearly identical EJ networks, suggesting that dehydration does not markedly alter the structure of this extracellular matrix.

Electron microscopy and forensic practice

NASA Astrophysics Data System (ADS)

Kotrlý, Marek; Turková, Ivana

2013-05-01

Electron microanalysis in forensic practice ranks among basic applications used in investigation of traces (latents, stains, etc.) from crime scenes. Applying electron microscope allows for rapid screening and receiving initial information for a wide range of traces. SEM with EDS/WDS makes it possible to observe topography surface and morphology samples and examination of chemical components. Physical laboratory of the Institute of Criminalistics Prague use SEM especially for examination of inorganic samples, rarely for biology and other material. Recently, possibilities of electron microscopy have been extended considerably using dual systems with focused ion beam. These systems are applied mainly in study of inner micro and nanoparticles , thin layers (intersecting lines in graphical forensic examinations, analysis of layers of functional glass, etc.), study of alloys microdefects, creating 3D particles and aggregates models, etc. Automated mineralogical analyses are a great asset to analysis of mineral phases, particularly soils, similarly it holds for cathode luminescence, predominantly colour one and precise quantitative measurement of their spectral characteristics. Among latest innovations that are becoming to appear also at ordinary laboratories are TOF - SIMS systems and micro Raman spectroscopy with a resolution comparable to EDS/WDS analysis (capable of achieving similar level as through EDS/WDS analysis).

Understanding materials challenges for rechargeable ion batteries with in situ transmission electron microscopy

NASA Astrophysics Data System (ADS)

Yuan, Yifei; Amine, Khalil; Lu, Jun; Shahbazian-Yassar, Reza

2017-08-01

An in-depth understanding of material behaviours under complex electrochemical environment is critical for the development of advanced materials for the next-generation rechargeable ion batteries. The dynamic conditions inside a working battery had not been intensively explored until the advent of various in situ characterization techniques. Real-time transmission electron microscopy of electrochemical reactions is one of the most significant breakthroughs poised to enable radical shift in our knowledge on how materials behave in the electrochemical environment. This review, therefore, summarizes the scientific discoveries enabled by in situ transmission electron microscopy, and specifically emphasizes the applicability of this technique to address the critical challenges in the rechargeable ion battery electrodes, electrolyte and their interfaces. New electrochemical systems such as lithium-oxygen, lithium-sulfur and sodium ion batteries are included, considering the rapidly increasing application of in situ transmission electron microscopy in these areas. A systematic comparison between lithium ion-based electrochemistry and sodium ion-based electrochemistry is also given in terms of their thermodynamic and kinetic differences. The effect of the electron beam on the validity of in situ observation is also covered. This review concludes by providing a renewed perspective for the future directions of in situ transmission electron microscopy in rechargeable ion batteries.

Understanding materials challenges for rechargeable ion batteries with in situ transmission electron microscopy

PubMed Central

Yuan, Yifei; Amine, Khalil; Lu, Jun; Shahbazian-Yassar, Reza

2017-01-01

An in-depth understanding of material behaviours under complex electrochemical environment is critical for the development of advanced materials for the next-generation rechargeable ion batteries. The dynamic conditions inside a working battery had not been intensively explored until the advent of various in situ characterization techniques. Real-time transmission electron microscopy of electrochemical reactions is one of the most significant breakthroughs poised to enable radical shift in our knowledge on how materials behave in the electrochemical environment. This review, therefore, summarizes the scientific discoveries enabled by in situ transmission electron microscopy, and specifically emphasizes the applicability of this technique to address the critical challenges in the rechargeable ion battery electrodes, electrolyte and their interfaces. New electrochemical systems such as lithium–oxygen, lithium–sulfur and sodium ion batteries are included, considering the rapidly increasing application of in situ transmission electron microscopy in these areas. A systematic comparison between lithium ion-based electrochemistry and sodium ion-based electrochemistry is also given in terms of their thermodynamic and kinetic differences. The effect of the electron beam on the validity of in situ observation is also covered. This review concludes by providing a renewed perspective for the future directions of in situ transmission electron microscopy in rechargeable ion batteries.

Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

NASA Astrophysics Data System (ADS)

Jesse, S.; Chi, M.; Belianinov, A.; Beekman, C.; Kalinin, S. V.; Borisevich, A. Y.; Lupini, A. R.

2016-05-01

Electron microscopy is undergoing a transition; from the model of producing only a few micrographs, through the current state where many images and spectra can be digitally recorded, to a new mode where very large volumes of data (movies, ptychographic and multi-dimensional series) can be rapidly obtained. Here, we discuss the application of so-called “big-data” methods to high dimensional microscopy data, using unsupervised multivariate statistical techniques, in order to explore salient image features in a specific example of BiFeO3 domains. Remarkably, k-means clustering reveals domain differentiation despite the fact that the algorithm is purely statistical in nature and does not require any prior information regarding the material, any coexisting phases, or any differentiating structures. While this is a somewhat trivial case, this example signifies the extraction of useful physical and structural information without any prior bias regarding the sample or the instrumental modality. Further interpretation of these types of results may still require human intervention. However, the open nature of this algorithm and its wide availability, enable broad collaborations and exploratory work necessary to enable efficient data analysis in electron microscopy.

Big Data Analytics for Scanning Transmission Electron Microscopy Ptychography

PubMed Central

Jesse, S.; Chi, M.; Belianinov, A.; Beekman, C.; Kalinin, S. V.; Borisevich, A. Y.; Lupini, A. R.

2016-01-01

Electron microscopy is undergoing a transition; from the model of producing only a few micrographs, through the current state where many images and spectra can be digitally recorded, to a new mode where very large volumes of data (movies, ptychographic and multi-dimensional series) can be rapidly obtained. Here, we discuss the application of so-called “big-data” methods to high dimensional microscopy data, using unsupervised multivariate statistical techniques, in order to explore salient image features in a specific example of BiFeO3 domains. Remarkably, k-means clustering reveals domain differentiation despite the fact that the algorithm is purely statistical in nature and does not require any prior information regarding the material, any coexisting phases, or any differentiating structures. While this is a somewhat trivial case, this example signifies the extraction of useful physical and structural information without any prior bias regarding the sample or the instrumental modality. Further interpretation of these types of results may still require human intervention. However, the open nature of this algorithm and its wide availability, enable broad collaborations and exploratory work necessary to enable efficient data analysis in electron microscopy. PMID:27211523

A fast image simulation algorithm for scanning transmission electron microscopy.

PubMed

Ophus, Colin

2017-01-01

Image simulation for scanning transmission electron microscopy at atomic resolution for samples with realistic dimensions can require very large computation times using existing simulation algorithms. We present a new algorithm named PRISM that combines features of the two most commonly used algorithms, namely the Bloch wave and multislice methods. PRISM uses a Fourier interpolation factor f that has typical values of 4-20 for atomic resolution simulations. We show that in many cases PRISM can provide a speedup that scales with f 4 compared to multislice simulations, with a negligible loss of accuracy. We demonstrate the usefulness of this method with large-scale scanning transmission electron microscopy image simulations of a crystalline nanoparticle on an amorphous carbon substrate.

Characterizing nanoscale scanning probes using electron microscopy: A novel fixture and a practical guide

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jacobs, Tevis D. B., E-mail: tjacobs@pitt.edu; Wabiszewski, Graham E.; Goodman, Alexander J.

2016-01-15

The nanoscale geometry of probe tips used for atomic force microscopy (AFM) measurements determines the lateral resolution, contributes to the strength of the tip-surface interaction, and can be a significant source of uncertainty in the quantitative analysis of results. While inverse imaging of the probe tip has been used successfully to determine probe tip geometry, direct observation of the tip profile using electron microscopy (EM) confers several advantages: it provides direct (rather than indirect) imaging, requires fewer algorithmic parameters, and does not require bringing the tip into contact with a sample. In the past, EM-based observation of the probe tipmore » has been achieved using ad hoc mounting methods that are constrained by low throughput, the risk of contamination, and repeatability issues. We report on a probe fixture designed for use in a commercial transmission electron microscope that enables repeatable mounting of multiple AFM probes as well as a reference grid for beam alignment. This communication describes the design, fabrication, and advantages of this probe fixture, including full technical drawings for machining. Further, best practices are discussed for repeatable, non-destructive probe imaging. Finally, examples of the fixture’s use are described, including characterization of common commercial AFM probes in their out-of-the-box condition.« less

Characterizing nanoscale scanning probes using electron microscopy: A novel fixture and a practical guide

NASA Astrophysics Data System (ADS)

Jacobs, Tevis D. B.; Wabiszewski, Graham E.; Goodman, Alexander J.; Carpick, Robert W.

2016-01-01

The nanoscale geometry of probe tips used for atomic force microscopy (AFM) measurements determines the lateral resolution, contributes to the strength of the tip-surface interaction, and can be a significant source of uncertainty in the quantitative analysis of results. While inverse imaging of the probe tip has been used successfully to determine probe tip geometry, direct observation of the tip profile using electron microscopy (EM) confers several advantages: it provides direct (rather than indirect) imaging, requires fewer algorithmic parameters, and does not require bringing the tip into contact with a sample. In the past, EM-based observation of the probe tip has been achieved using ad hoc mounting methods that are constrained by low throughput, the risk of contamination, and repeatability issues. We report on a probe fixture designed for use in a commercial transmission electron microscope that enables repeatable mounting of multiple AFM probes as well as a reference grid for beam alignment. This communication describes the design, fabrication, and advantages of this probe fixture, including full technical drawings for machining. Further, best practices are discussed for repeatable, non-destructive probe imaging. Finally, examples of the fixture's use are described, including characterization of common commercial AFM probes in their out-of-the-box condition.

Scanning electron microscopy of superficial white onychomycosis*

PubMed Central

de Almeida Jr., Hiram Larangeira; Boabaid, Roberta Oliveira; Timm, Vitor; Silva, Ricardo Marques e; de Castro, Luis Antonio Suita

2015-01-01

Superficial white onychomycosis is characterized by opaque, friable, whitish superficial spots on the nail plate. We examined an affected halux nail of a 20-year-old male patient with scanning electron microscopy. The mycological examination isolated Trichophyton mentagrophytes. Abundant hyphae with the formation of arthrospores were found on the nail's surface, forming small fungal colonies. These findings showed the great capacity for dissemination of this form of onychomycosis. PMID:26560225

System and method for compressive scanning electron microscopy

DOEpatents

Reed, Bryan W

2015-01-13

A scanning transmission electron microscopy (STEM) system is disclosed. The system may make use of an electron beam scanning system configured to generate a plurality of electron beam scans over substantially an entire sample, with each scan varying in electron-illumination intensity over a course of the scan. A signal acquisition system may be used for obtaining at least one of an image, a diffraction pattern, or a spectrum from the scans, the image, diffraction pattern, or spectrum representing only information from at least one of a select subplurality or linear combination of all pixel locations comprising the image. A dataset may be produced from the information. A subsystem may be used for mathematically analyzing the dataset to predict actual information that would have been produced by each pixel location of the image.

Analysis of Peroxisome Biogenesis in Pollen by Confocal Microscopy and Transmission Electron Microscopy.

PubMed

Jia, Peng-Fei; Li, Hong-Ju; Yang, Wei-Cai

2017-01-01

Peroxisome is an essential single-membrane bound organelle in most eukaryotic cells and functions in diverse cellular processes. De novo formation, division, and turnover of peroxisomes contribute to its biogenesis, morphology, and population regulation. In plants, peroxisome plays multiple roles, including metabolism, development, and stress response. Defective peroxisome biogenesis and development retard plant growth, adaption, and reproduction. Through tracing the subcellular localization of fluorescent reporter tagged matrix protein of peroxisome, fluorescence microscopy is a reliable and fast way to detect peroxisome biogenesis. Further fine-structural observation of peroxisome by TEM enables researchers to observe the detailed ultrastructure of its morphology and spatial contact with other organelles. Pollen grain is a specialized structure where two small sperm cells are enclosed in the cytoplasm of a large vegetative cell. Two features make pollen grain a good system to study peroxisome biogenesis: indispensable requirement of peroxisome for germination on the stigma and homogeneity. Here, we describe the methods of studying peroxisome biogenesis in Arabidopsis pollen grains by fluorescent live-imaging with confocal laser scanning microscopy (CLSM) and by DAB-staining based transmission electron microscopy (TEM).

Advanced electron microscopy methods for the analysis of MgB2 superconductor

NASA Astrophysics Data System (ADS)

Birajdar, B.; Peranio, N.; Eibl, O.

2008-02-01

Advanced electron microscopy methods used for the analysis of superconducting MgB2 wires and tapes are described. The wires and tapes were prepared by the powder in tube method using different processing technologies and thoroughly characterised for their superconducting properties within the HIPERMAG project. Microstructure analysis on μm to nm length scales is necessary to understand the superconducting properties of MgB2. For the MgB2 phase analysis on μm scale an analytical SEM, and for the analysis on nm scale a energy-filtered STEM is used. Both the microscopes were equipped with EDX detector and field emission gun. Electron microscopy and spectroscopy of MgB2 is challenging because of the boron analysis, carbon and oxygen contamination, and the presence of large number of secondary phases. Advanced electron microscopy involves, combined SEM, EPMA and TEM analysis with artefact free sample preparation, elemental mapping and chemical quantification of point spectra. Details of the acquisition conditions and achieved accuracy are presented. Ex-situ wires show oxygen-free MgB2 colonies (a colony is a dense arrangement of several MgB2 grains) embedded in a porous and oxygen-rich matrix, introducing structural granularity. In comparison, in-situ wires are generally more dense, but show inhibited MgB2 phase formation with significantly higher fraction of B-rich secondary phases. SiC additives in the in-situ wires forms Mg2Si secondary phases. The advanced electron microscopy has been used to extract the microstructure parameters like colony size, B-rich secondary phase fraction, O mole fraction and MgB2 grain size, and establish a microstructure-critical current density model [1]. In summary, conventional secondary electron imaging in SEM and diffraction contrast imaging in the TEM are by far not sufficient and advanced electron microscopy methods are essential for the analysis of superconducting MgB2 wires and tapes.

Ultrafast electron microscopy in materials science, biology, and chemistry

NASA Astrophysics Data System (ADS)

King, Wayne E.; Campbell, Geoffrey H.; Frank, Alan; Reed, Bryan; Schmerge, John F.; Siwick, Bradley J.; Stuart, Brent C.; Weber, Peter M.

2005-06-01

The use of pump-probe experiments to study complex transient events has been an area of significant interest in materials science, biology, and chemistry. While the emphasis has been on laser pump with laser probe and laser pump with x-ray probe experiments, there is a significant and growing interest in using electrons as probes. Early experiments used electrons for gas-phase diffraction of photostimulated chemical reactions. More recently, scientists are beginning to explore phenomena in the solid state such as phase transformations, twinning, solid-state chemical reactions, radiation damage, and shock propagation. This review focuses on the emerging area of ultrafast electron microscopy (UEM), which comprises ultrafast electron diffraction (UED) and dynamic transmission electron microscopy (DTEM). The topics that are treated include the following: (1) The physics of electrons as an ultrafast probe. This encompasses the propagation dynamics of the electrons (space-charge effect, Child's law, Boersch effect) and extends to relativistic effects. (2) The anatomy of UED and DTEM instruments. This includes discussions of the photoactivated electron gun (also known as photogun or photoelectron gun) at conventional energies (60-200 keV) and extends to MeV beams generated by rf guns. Another critical aspect of the systems is the electron detector. Charge-coupled device cameras and microchannel-plate-based cameras are compared and contrasted. The effect of various physical phenomena on detective quantum efficiency is discussed. (3) Practical aspects of operation. This includes determination of time zero, measurement of pulse-length, and strategies for pulse compression. (4) Current and potential applications in materials science, biology, and chemistry. UEM has the potential to make a significant impact in future science and technology. Understanding of reaction pathways of complex transient phenomena in materials science, biology, and chemistry will provide fundamental

Simplifying Electron Beam Channeling in Scanning Transmission Electron Microscopy (STEM).

PubMed

Wu, Ryan J; Mittal, Anudha; Odlyzko, Michael L; Mkhoyan, K Andre

2017-08-01

Sub-angstrom scanning transmission electron microscopy (STEM) allows quantitative column-by-column analysis of crystalline specimens via annular dark-field images. The intensity of electrons scattered from a particular location in an atomic column depends on the intensity of the electron probe at that location. Electron beam channeling causes oscillations in the STEM probe intensity during specimen propagation, which leads to differences in the beam intensity incident at different depths. Understanding the parameters that control this complex behavior is critical for interpreting experimental STEM results. In this work, theoretical analysis of the STEM probe intensity reveals that intensity oscillations during specimen propagation are regulated by changes in the beam's angular distribution. Three distinct regimes of channeling behavior are observed: the high-atomic-number (Z) regime, in which atomic scattering leads to significant angular redistribution of the beam; the low-Z regime, in which the probe's initial angular distribution controls intensity oscillations; and the intermediate-Z regime, in which the behavior is mixed. These contrasting regimes are shown to exist for a wide range of probe parameters. These results provide a new understanding of the occurrence and consequences of channeling phenomena and conditions under which their influence is strengthened or weakened by characteristics of the electron probe and sample.

X-ray microscopy as an approach to increasing accuracy and efficiency of serial block-face imaging for correlated light and electron microscopy of biological specimens.

PubMed

Bushong, Eric A; Johnson, Donald D; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H

2015-02-01

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging.

X-ray Microscopy as an Approach to Increasing Accuracy and Efficiency of Serial Block-face Imaging for Correlated Light and Electron Microscopy of Biological Specimens

PubMed Central

Bushong, Eric A.; Johnson, Donald D.; Kim, Keun-Young; Terada, Masako; Hatori, Megumi; Peltier, Steven T.; Panda, Satchidananda; Merkle, Arno; Ellisman, Mark H.

2015-01-01

The recently developed three-dimensional electron microscopic (EM) method of serial block-face scanning electron microscopy (SBEM) has rapidly established itself as a powerful imaging approach. Volume EM imaging with this scanning electron microscopy (SEM) method requires intense staining of biological specimens with heavy metals to allow sufficient back-scatter electron signal and also to render specimens sufficiently conductive to control charging artifacts. These more extreme heavy metal staining protocols render specimens light opaque and make it much more difficult to track and identify regions of interest (ROIs) for the SBEM imaging process than for a typical thin section transmission electron microscopy correlative light and electron microscopy study. We present a strategy employing X-ray microscopy (XRM) both for tracking ROIs and for increasing the efficiency of the workflow used for typical projects undertaken with SBEM. XRM was found to reveal an impressive level of detail in tissue heavily stained for SBEM imaging, allowing for the identification of tissue landmarks that can be subsequently used to guide data collection in the SEM. Furthermore, specific labeling of individual cells using diaminobenzidine is detectable in XRM volumes. We demonstrate that tungsten carbide particles or upconverting nanophosphor particles can be used as fiducial markers to further increase the precision and efficiency of SBEM imaging. PMID:25392009

The Electron Microscopy Outreach Program: A Web-based resource for research and education.

PubMed

Sosinsky, G E; Baker, T S; Hand, G; Ellisman, M H

1999-01-01

We have developed a centralized World Wide Web (WWW)-based environment that serves as a resource of software tools and expertise for biological electron microscopy. A major focus is molecular electron microscopy, but the site also includes information and links on structural biology at all levels of resolution. This site serves to help integrate or link structural biology techniques in accordance with user needs. The WWW site, called the Electron Microscopy (EM) Outreach Program (URL: http://emoutreach.sdsc.edu), provides scientists with computational and educational tools for their research and edification. In particular, we have set up a centralized resource containing course notes, references, and links to image analysis and three-dimensional reconstruction software for investigators wanting to learn about EM techniques either within or outside of their fields of expertise. Copyright 1999 Academic Press.

A fast image simulation algorithm for scanning transmission electron microscopy

DOE PAGES

Ophus, Colin

2017-05-10

Image simulation for scanning transmission electron microscopy at atomic resolution for samples with realistic dimensions can require very large computation times using existing simulation algorithms. Here, we present a new algorithm named PRISM that combines features of the two most commonly used algorithms, namely the Bloch wave and multislice methods. PRISM uses a Fourier interpolation factor f that has typical values of 4-20 for atomic resolution simulations. We show that in many cases PRISM can provide a speedup that scales with f 4 compared to multislice simulations, with a negligible loss of accuracy. We demonstrate the usefulness of this methodmore » with large-scale scanning transmission electron microscopy image simulations of a crystalline nanoparticle on an amorphous carbon substrate.« less

Domain imaging in ferroelectric thin films via channeling-contrast backscattered electron microscopy

DOE PAGES

Ihlefeld, Jon F.; Michael, Joseph R.; McKenzie, Bonnie B.; ...

2016-09-16

We report that ferroelastic domain walls provide opportunities for deterministically controlling mechanical, optical, electrical, and thermal energy. Domain wall characterization in micro- and nanoscale systems, where their spacing may be of the order of 100 nm or less is presently limited to only a few techniques, such as piezoresponse force microscopy and transmission electron microscopy. These respective techniques cannot, however, independently characterize domain polarization orientation and domain wall motion in technologically relevant capacitor structures or in a non-destructive manner, thus presenting a limitation of their utility. In this work, we show how backscatter scanning electron microscopy utilizing channeling contrast yieldmore » can image the ferroelastic domain structure of ferroelectric films with domain wall spacing as narrow as 10 nm.« less

Probing cytotoxicity of nanoparticles and organic compounds using scanning proton microscopy, scanning electron microscopy and fluorescence microscopy

NASA Astrophysics Data System (ADS)

Tong, Yongpeng; Li, Changming; Liang, Feng; Chen, Jianmin; Zhang, Hong; Liu, Guoqing; Sun, Huibin; Luong, John H. T.

2008-12-01

Scanning proton microscopy, scanning electron microscopy (SEM) and fluorescence microscopy have been used to probe the cytotoxicity effect of benzo[a]pyrene (BaP), ethidium bromide (EB) and nanoparticles (ZnO, Al 2O 3 and TiO 2) on a T lymphoblastic leukemia Jurkat cell line. The increased calcium ion (from CaCl 2) in the culture medium stimulated the accumulation of BaP and EB inside the cell, leading to cell death. ZnO, Al 2O 3 and TiO 2 nanoparticles, however, showed a protective effect against these two organic compounds. Such inorganic nanoparticles complexed with BaP or EB which became less toxic to the cell. Fe 2O 3 nanoparticles as an insoluble particle model scavenged by macrophage were investigated in rats. They were scavenged out of the lung tissue about 48 h after infection. This result suggest that some insoluble inorganic nanoparticles of PM (particulate matters) showed protective effects on organic toxins induced acute toxic effects as they can be scavenged by macrophage cells. Whereas, some inorganic ions such as calcium ion in PM may help environmental organic toxins to penetrate cell membrane and induce higher toxic effect.

Resinless section electron microscopy reveals the yeast cytoskeleton.

PubMed

Penman, J; Penman, S

1997-04-15

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or "soluble" proteins are distinct from the retained or "structural" proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters-5 nm and 15-20 nm-which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300-500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture.

Electron microscopy observations of radiation damage in irradiated and annealed tungsten

NASA Astrophysics Data System (ADS)

Grzonka, J.; Ciupiński, Ł.; Smalc-Koziorowska, J.; Ogorodnikova, O. V.; Mayer, M.; Kurzydłowski, K. J.

2014-12-01

In the present work tungsten samples were irradiated with W6+ ions with a kinetic energy of 20 MeV in order to simulate radiation damage by fast neutrons. Two samples with cumulative damage of 2.3 and 6.36 displacements per atom were produced. The scanning transmission electron microscopy investigations were carried out in order to determine structure changes resulting from the irradiation. The evolution of the damage with post implantation annealing in the temperature range 673-1100 K was also assessed. Damage profiles were studied at cross-sections. Scanning transmission electron microscopy studies of the lamellae after annealing revealed aggregation of defects and rearrangement as well as partial healing of dislocations at higher temperatures. The results confirm the higher density of radiation-induced dislocations in the near surface area of the sample (1.8 * 1014 m-2) in comparison with a deeper damage area (1.5 * 1014 m-2). Significant decrease of dislocation density was observed after annealing with a concurrent growth of dislocation loops. Transmission electron microscopy analyses show that the dislocation loops are perfect dislocations with the Burgers vectors of b = ½[ 1 1 1].

High-resolution scanning electron microscopy of frozen-hydrated cells.

PubMed

Walther, P; Chen, Y; Pech, L L; Pawley, J B

1992-11-01

Cryo-fixed yeast Paramecia and sea urchin embryos were investigated with an in-lens type field-emission SEM using a cold stage. The goal was to further develop and investigate the processing of frozen samples for the low-temperature scanning electron microscope (LTSEM). Uncoated frozen-hydrated samples were imaged with the low-voltage backscattered electron signal (BSE). Resolution and contrast were sufficient to visualize cross-fractured membranes, nuclear pores and small vesicles in the cytoplasm. It is assumed that the resolution of this approach is limited by the extraction depth of the BSE which depends upon the accelerating voltage of the primary beam (V0). In this study, the lowest possible V0 was 2.6 kV because below this value the sensitivity of the BSE detector is insufficient. It is concluded that the resolution of the uncoated specimen could be improved if equipment were available for high-resolution BSE imaging at 0.5-2 kV. Higher resolution was obtained with platinum cryo-coated samples, on which intramembranous particles were easily imaged. These images even show the ring-like appearance of the hexagonally arranged intramembranous particles known from high-resolution replica studies. On fully hydrated samples at high magnification, the observation time for a particular area is limited by mass loss caused by electron irradiation. Other potential sources of artefacts are the deposition of water vapour contamination and shrinkage caused by the sublimation of ice. Imaging of partially dehydrated (partially freeze-dried) samples, e.g. high-pressure frozen Paramecium and sea urchin embryos, will probably become the main application in cell biology. In spite of possible shrinkage problems, this approach has a number of advantages compared with any other electron microscopy preparation method: no chemical fixation is necessary, eliminating this source of artefacts; due to partial removal of the water additional structures in the cytoplasm can be investigated

A novel cell culture technique for electron microscopy.

PubMed

Wang, F; Ledford, L B; Head, J F; Elliott, R L

1993-12-15

A simplified technique for the monolayer growth of cultured cells and their in situ embedment on the inner surface of the pyramidal portion of the Beem capsule for electron microscopy has been developed. The results demonstrated that the cell monolayers grew well on the surface of the Beem capsule and could be embedded in situ. Electron micrographs showed cells in their natural state of contact with one another. The plasma membrane and intracellular organelles were well preserved. This method minimizes many difficult steps and eliminates the disruption of cells by scraping, pelleting, or enzymatic reaction to remove them.

A Survey of the Use of Iterative Reconstruction Algorithms in Electron Microscopy

PubMed Central

Otón, J.; Vilas, J. L.; Kazemi, M.; Melero, R.; del Caño, L.; Cuenca, J.; Conesa, P.; Gómez-Blanco, J.; Marabini, R.; Carazo, J. M.

2017-01-01

One of the key steps in Electron Microscopy is the tomographic reconstruction of a three-dimensional (3D) map of the specimen being studied from a set of two-dimensional (2D) projections acquired at the microscope. This tomographic reconstruction may be performed with different reconstruction algorithms that can be grouped into several large families: direct Fourier inversion methods, back-projection methods, Radon methods, or iterative algorithms. In this review, we focus on the latter family of algorithms, explaining the mathematical rationale behind the different algorithms in this family as they have been introduced in the field of Electron Microscopy. We cover their use in Single Particle Analysis (SPA) as well as in Electron Tomography (ET). PMID:29312997

Cryo-electron microscopy of membrane proteins.

PubMed

Goldie, Kenneth N; Abeyrathne, Priyanka; Kebbel, Fabian; Chami, Mohamed; Ringler, Philippe; Stahlberg, Henning

2014-01-01

Electron crystallography is used to study membrane proteins in the form of planar, two-dimensional (2D) crystals, or other crystalline arrays such as tubular crystals. This method has been used to determine the atomic resolution structures of bacteriorhodopsin, tubulin, aquaporins, and several other membrane proteins. In addition, a large number of membrane protein structures were studied at a slightly lower resolution, whereby at least secondary structure motifs could be identified.In order to conserve the structural details of delicate crystalline arrays, cryo-electron microscopy (cryo-EM) allows imaging and/or electron diffraction of membrane proteins in their close-to-native state within a lipid bilayer membrane.To achieve ultimate high-resolution structural information of 2D crystals, meticulous sample preparation for electron crystallography is of outmost importance. Beam-induced specimen drift and lack of specimen flatness can severely affect the attainable resolution of images for tilted samples. Sample preparations that sandwich the 2D crystals between symmetrical carbon films reduce the beam-induced specimen drift, and the flatness of the preparations can be optimized by the choice of the grid material and the preparation protocol.Data collection in the cryo-electron microscope using either the imaging or the electron diffraction mode has to be performed applying low-dose procedures. Spot-scanning further reduces the effects of beam-induced drift. Data collection using automated acquisition schemes, along with improved and user-friendlier data processing software, is increasingly being used and is likely to bring the technique to a wider user base.

Retracing in correlative light electron microscopy: where is my object of interest?

PubMed

Hodgson, Lorna; Nam, David; Mantell, Judith; Achim, Alin; Verkade, Paul

2014-01-01

Correlative light electron microscopy (CLEM) combines the strengths of light and electron microscopy in a single experiment. There are many ways to perform a CLEM experiment and a variety of microscopy modalities can be combined either on separate instruments or as completely integrated solutions. In general, however, a CLEM experiment can be divided into three parts: probes, processing, and analysis. Most of the existing technologies are focussed around the development and use of probes or describe processing methodologies that explain or circumvent some of the compromises that need to be made when performing both light and electron microscopy on the same sample. So far, relatively little attention has been paid to the analysis part of CLEM experiments. Although it is an essential part of each CLEM experiment, it is usually a cumbersome manual process. Here, we briefly discuss each of the three above-mentioned steps, with a focus on the analysis part. We will also introduce an automated registration algorithm that can be applied to the analysis stage to enable the accurate registration of LM and EM images. This facilitates tracing back the right cell/object seen in the light microscope in the EM. © 2014 Elsevier Inc. All rights reserved.

Carbon contamination in scanning transmission electron microscopy and its impact on phase-plate applications.

PubMed

Hettler, Simon; Dries, Manuel; Hermann, Peter; Obermair, Martin; Gerthsen, Dagmar; Malac, Marek

2017-05-01

We analyze electron-beam induced carbon contamination in a transmission electron microscope. The study is performed on thin films potentially suitable as phase plates for phase-contrast transmission electron microscopy. Electron energy-loss spectroscopy and phase-plate imaging is utilized to analyze the contamination. The deposited contamination layer is identified as a graphitic carbon layer which is not prone to electrostatic charging whereas a non-conductive underlying substrate charges. Several methods that inhibit contamination are evaluated and the impact of carbon contamination on phase-plate imaging is discussed. The findings are in general interesting for scanning transmission electron microscopy applications. Crown Copyright © 2017. Published by Elsevier Ltd. All rights reserved.

Second harmonic generation quantitative measurements on collagen fibrils through correlation to electron microscopy

NASA Astrophysics Data System (ADS)

Bancelin, S.; Aimé, C.; Gusachenko, I.; Kowalczuk, L.; Latour, G.; Coradin, T.; Schanne-Klein, M.-C.

2015-03-01

Type I collagen is a major structural protein in mammals that shows highly structured macromolecular organizations specific to each tissue. This biopolymer is synthesized as triple helices, which self-assemble into fibrils (Ø =10-300 nm) and further form various 3D organization. In recent years, Second Harmonic Generation (SHG) microscopy has emerged as a powerful technique to probe in situ the fibrillar collagenous network within tissues. However, this optical technique cannot resolve most of the fibrils and is a coherent process, which has impeded quantitative measurements of the fibril diameter so far. In this study, we correlated SHG microscopy with Transmission Electron Microscopy to determine the sensitivity of SHG microscopy and to calibrate SHG signals as a function of the fibril diameter in reconstructed collagen gels. To that end, we synthetized isolated fibrils with various diameters and successfully imaged the very same fibrils with both techniques, down to 30 nm diameter. We observed that SHG signals scaled as the fourth power of the fibril diameter, as expected from analytical and numerical calculations. This calibration was then applied to diabetic rat cornea in which we successfully recovered the diameter of hyperglycemia-induced fibrils in the Descemet's membrane without having to resolve them. Finally we derived the first hyperpolarizability from a single collagen triple helix which validates the bottom-up approach used to calculate the non-linear response at the fibrillar scale and denotes a parallel alignment of triple helices within the fibrils. These results represent a major step towards quantitative SHG imaging of nm-sized collagen fibrils.

The Application of Electron Microscopy Techniques to the Space Shuttle Columbia Accident Investigation

NASA Technical Reports Server (NTRS)

Shah, Sandeep; Jerman, Greg

2005-01-01

The Space Shuttle Columbia was returning from a 16-day research mission, STS- 107, with nominal system performance prior to the beginning of the entry interface into earth's upper atmosphere. Approximately one minute and twenty four seconds into the peak heating region of the entry interface, an off-nominal temperature rise was observed in the left main landing gear brake line. Nearly seven minutes later, all contact was lost with Columbia. Debris was observed periodically exiting the Shuttle's flight path throughout the reentry profile over California, Nevada, and New Mexico, until its final breakup over Texas. During the subsequent investigation, electron microscopy techniques were crucial in revealing the location of the fatal damage that resulted in the loss of Columbia and her crew.

High-resolution electron microscopy and electron energy-loss spectroscopy of giant palladium clusters

NASA Astrophysics Data System (ADS)

Oleshko, V.; Volkov, V.; Gijbels, R.; Jacob, W.; Vargaftik, M.; Moiseev, I.; van Tendeloo, G.

1995-12-01

Combined structural and chemical characterization of cationic polynuclear palladium coordination compounds Pd561L60(OAc)180, where L=1,10-phenantroline or 2,2'-bipyridine has been carried out by high-resolution electron microscopy (HREM) and analytical electron microscopy methods including electron energy-loss spectroscopy (EELS), zero-loss electron spectroscopic imaging, and energy-dispersive X-ray spectroscopy (EDX). The cell structure of the cluster matter with almost completely uniform metal core size distributions centered around 2.3 ±0.5 nm was observed. Zero-loss energy filtering allowed to improve the image contrast and resolution. HREM images showed that most of the palladium clusters had a cubo-octahedral shape. Some of them had a distorted icosahedron structure exhibiting multiple twinning. The selected-area electron diffraction patterns confirmed the face centered cubic structure with lattice parameter close to that of metallic palladium. The energy-loss spectra of the populations of clusters contained several bands, which could be assigned to the delayed Pd M4, 5-edge at 362 eV, the Pd M3-edge at 533 eV and the Pd M2-edge at 561 eV, the NK-edge at about 400 eV, the O K-edge at 532 eV overlapping with the Pd M3-edge and the carbon C K-edge at 284 eV. Background subtraction was applied to reveal the exact positions and fine structure of low intensity elemental peaks. EELS evaluations have been confirmed by EDX. The recorded series of the Pd M-edges and the N K-edge in the spectra of the giant palladium clusters obviously were related to Pd-Pd- and Pd-ligand bonding.

Electron microscopy localization and characterization of functionalized composite organic-inorganic SERS nanoparticles on leukemia cells.

PubMed

Koh, Ai Leen; Shachaf, Catherine M; Elchuri, Sailaja; Nolan, Garry P; Sinclair, Robert

2008-12-01

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic nanoparticle (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet scanning electron microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron (BSE) detector was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution transmission electron microscopy (TEM) images and scanning Auger electron spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens.

Electron microscopy characterization of Ni-Cr-B-Si-C laser deposited coatings.

PubMed

Hemmati, I; Rao, J C; Ocelík, V; De Hosson, J Th M

2013-02-01

During laser deposition of Ni-Cr-B-Si-C alloys with high amounts of Cr and B, various microstructures and phases can be generated from the same chemical composition that results in heterogeneous properties in the clad layer. In this study, the microstructure and phase constitution of a high-alloy Ni-Cr-B-Si-C coating deposited by laser cladding were analyzed by a combination of several microscopy characterization techniques including scanning electron microscopy in secondary and backscatter imaging modes, energy dispersive spectroscopy (EDS), electron backscatter diffraction (EBSD), and transmission electron microscopy (TEM). The combination of EDS and EBSD allowed unequivocal identification of micron-sized precipitates as polycrystalline orthorhombic CrB, single crystal tetragonal Cr5B3, and single crystal hexagonal Cr7C3. In addition, TEM characterization showed various equilibrium and metastable Ni-B, Ni-Si, and Ni-Si-B eutectic products in the alloy matrix. The findings of this study can be used to explain the phase formation reactions and to tune the microstructure of Ni-Cr-B-Si-C coatings to obtain the desired properties.

Low-temperature and conventional scanning electron microscopy of human urothelial neoplasms.

PubMed

Hopkins, D M; Morris, J A; Oates, K; Huddart, H; Staff, W G

1989-05-01

The appearance of neoplastic human urothelium viewed by low-temperature scanning electron microscopy (LTSEM) and conventional scanning electron microscopy (CSEM) was compared. Fixed, dehydrated neoplastic cells viewed by CSEM had well-defined, often raised cell junctions; no intercellular gaps; and varying degrees of pleomorphic surface microvilli. The frozen hydrated material viewed by LTSEM, however, was quite different. The cells had a flat or dimpled surface, but no microvilli. There were labyrinthine lateral processes which interdigitated with those of adjacent cells and outlined large intercellular gaps. The process of fixation and dehydration will inevitably distort cell contours and on theoretical grounds, the images of frozen hydrated material should more closely resemble the in vivo appearance.

Electron microscopy study of gold nanoparticles deposited on transition metal oxides.

PubMed

Akita, Tomoki; Kohyama, Masanori; Haruta, Masatake

2013-08-20

Many researchers have investigated the catalytic performance of gold nanoparticles (GNPs) supported on metal oxides for various catalytic reactions of industrial importance. These studies have consistently shown that the catalytic activity and selectivity depend on the size of GNPs, the kind of metal oxide supports, and the gold/metal oxide interface structure. Although researchers have proposed several structural models for the catalytically active sites and have identified the specific electronic structures of GNPs induced by the quantum effect, recent experimental and theoretical studies indicate that the perimeter around GNPs in contact with the metal oxide supports acts as an active site in many reactions. Thus, it is of immense importance to investigate the detailed structures of the perimeters and the contact interfaces of gold/metal oxide systems by using electron microscopy at an atomic scale. This Account describes our investigation, at the atomic scale using electron microscopy, of GNPs deposited on metal oxides. In particular, high-resolution transmission electron microscopy (HRTEM) and high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) are valuable tools to observe local atomic structures, as has been successfully demonstrated for various nanoparticles, surfaces, and material interfaces. TEM can be applied to real powder catalysts as received without making special specimens, in contrast to what is typically necessary to observe bulk materials. For precise structure analyses at an atomic scale, model catalysts prepared by using well-defined single-crystalline substrates are also adopted for TEM observations. Moreover, aberration-corrected TEM, which has high spatial resolution under 0.1 nm, is a promising tool to observe the interface structure between GNPs and metal oxide supports including oxygen atoms at the interfaces. The oxygen atoms in particular play an important role in the behavior of gold/metal oxide

Correlative Light- and Electron Microscopy Using Quantum Dot Nanoparticles.

PubMed

Killingsworth, Murray C; Bobryshev, Yuri V

2016-08-07

A method is described whereby quantum dot (QD) nanoparticles can be used for correlative immunocytochemical studies of human pathology tissue using widefield fluorescence light microscopy and transmission electron microscopy (TEM). To demonstrate the protocol we have immunolabeled ultrathin epoxy sections of human somatostatinoma tumor using a primary antibody to somatostatin, followed by a biotinylated secondary antibody and visualization with streptavidin conjugated 585 nm cadmium-selenium (CdSe) quantum dots (QDs). The sections are mounted on a TEM specimen grid then placed on a glass slide for observation by widefield fluorescence light microscopy. Light microscopy reveals 585 nm QD labeling as bright orange fluorescence forming a granular pattern within the tumor cell cytoplasm. At low to mid-range magnification by light microscopy the labeling pattern can be easily recognized and the level of non-specific or background labeling assessed. This is a critical step for subsequent interpretation of the immunolabeling pattern by TEM and evaluation of the morphological context. The same section is then blotted dry and viewed by TEM. QD probes are seen to be attached to amorphous material contained in individual secretory granules. Images are acquired from the same region of interest (ROI) seen by light microscopy for correlative analysis. Corresponding images from each modality may then be blended to overlay fluorescence data on TEM ultrastructure of the corresponding region.

Ultrafast Imaging of Chiral Surface Plasmon by Photoemission Electron Microscopy

NASA Astrophysics Data System (ADS)

Dai, Yanan; Dabrowski, Maciej; Petek, Hrvoje

We employ Time-Resolved Photoemission Electron Microscopy (TR-PEEM) to study surface plasmon polariton (SPP) wave packet dynamics launched by tunable (VIS-UV) femtosecond pulses of various linear and circular polarizations. The plasmonic structures are micron size single-crystalline Ag islands grown in situ on Si surfaces and characterized by Low Energy Electron Microscopy (LEEM). The local fields of plasmonic modes enhance two and three photon photoemission (2PP and 3PP) at the regions of strong field enhancement. Imaging of the photoemission signal with PEEM electron optics thus images the plasmonic fields excited in the samples. The observed PEEM images with left and right circularly polarized light show chiral images, which is a consequence of the transverse spin momentum of surface plasmon. By changing incident light polarization, the plasmon interference pattern shifts with light ellipticity indicating a polarization dependent excitation phase of SPP. In addition, interferometric-time resolved measurements record the asymmetric SPP wave packet motion in order to characterize the dynamical properties of chiral SPP wave packets.

Resinless section electron microscopy reveals the yeast cytoskeleton

PubMed Central

Penman, Joshua; Penman, Sheldon

1997-01-01

The cytoskeleton of Saccharomyces cerevisiae is essentially invisible using conventional microscopy techniques. A similar problem was solved for the mammalian cell cytoskeleton using resinless section electron microscopy, a technique applied here to yeast. In the resinless image, soluble proteins are no longer cloaked by embedding medium and must be removed by selective detergent extraction. In yeast, this requires breaching the cell wall by digesting with Zymolyase sufficiently to allow detergent extraction of the plasma membrane lipids. Gel electropherograms show that the extracted or “soluble” proteins are distinct from the retained or “structural” proteins that presumably comprise the cytoskeleton. These putative cytoskeleton proteins include the major portions of a 43-kDa protein, which is presumably actin, and of proteins in a band appearing at 55 kDa, as well as numerous less abundant, nonactin proteins. Resinless section electron micrographs show a dense, three-dimensional web of anastomosing, polymorphic filaments bounded by the remnant cell wall. Although the filament network is very heterogenous, there appear to be two principal classes of filament diameters—5 nm and 15–20 nm—which may correspond to actin and intermediate filaments, respectively. A large oval region of lower filament density probably corresponds to the vacuole, and an electron dense spheroidal body, 300–500 nm in diameter, is likely the nucleus. The techniques detailed in this report afford new approaches to the study of yeast cytoarchitecture. PMID:9108046

Electron and Fluorescence Microscopy of Extracellular Glucan and Aryl-Alcohol Oxidase during Wheat-Straw Degradation by Pleurotus eryngii

PubMed Central

Barrasa, J. M.; Gutiérrez, A.; Escaso, V.; Guillén, F.; Martínez, M. J.; Martínez, A. T.

1998-01-01

The ligninolytic fungus Pleurotus eryngii grown in liquid medium secreted extracellular polysaccharide (87% glucose) and the H2O2-producing enzyme aryl-alcohol oxidase (AAO). The production of both was stimulated by wheat-straw. Polyclonal antibodies against purified AAO were obtained, and a complex of glucanase and colloidal gold was prepared. With these tools, the localization of AAO and extracellular glucan in mycelium from liquid medium and straw degraded under solid-state fermentation conditions was investigated by transmission electron microscopy (TEM) and fluorescence microscopy. These studies revealed that P. eryngii produces a hyphal sheath consisting of a thin glucan layer. This sheath appeared to be involved in both mycelial adhesion to the straw cell wall during degradation and AAO immobilization on hyphal surfaces, with the latter evidenced by double labeling. AAO distribution during differential degradation of straw tissues was observed by immunofluorescence microscopy. Finally, TEM immunogold studies confirmed that AAO penetrates the plant cell wall during P. eryngii degradation of wheat straw. PMID:9435085

Accurate virus quantitation using a Scanning Transmission Electron Microscopy (STEM) detector in a scanning electron microscope.

PubMed

Blancett, Candace D; Fetterer, David P; Koistinen, Keith A; Morazzani, Elaine M; Monninger, Mitchell K; Piper, Ashley E; Kuehl, Kathleen A; Kearney, Brian J; Norris, Sarah L; Rossi, Cynthia A; Glass, Pamela J; Sun, Mei G

2017-10-01

A method for accurate quantitation of virus particles has long been sought, but a perfect method still eludes the scientific community. Electron Microscopy (EM) quantitation is a valuable technique because it provides direct morphology information and counts of all viral particles, whether or not they are infectious. In the past, EM negative stain quantitation methods have been cited as inaccurate, non-reproducible, and with detection limits that were too high to be useful. To improve accuracy and reproducibility, we have developed a method termed Scanning Transmission Electron Microscopy - Virus Quantitation (STEM-VQ), which simplifies sample preparation and uses a high throughput STEM detector in a Scanning Electron Microscope (SEM) coupled with commercially available software. In this paper, we demonstrate STEM-VQ with an alphavirus stock preparation to present the method's accuracy and reproducibility, including a comparison of STEM-VQ to viral plaque assay and the ViroCyt Virus Counter. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

Characterization of Sulfur and Nanostructured Sulfur Battery Cathodes in Electron Microscopy Without Sublimation Artifacts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Levin, Barnaby D. A.; Zachman, Michael J.; Werner, Jörg G.

Abstract Lithium sulfur (Li–S) batteries have the potential to provide higher energy storage density at lower cost than conventional lithium ion batteries. A key challenge for Li–S batteries is the loss of sulfur to the electrolyte during cycling. This loss can be mitigated by sequestering the sulfur in nanostructured carbon–sulfur composites. The nanoscale characterization of the sulfur distribution within these complex nanostructured electrodes is normally performed by electron microscopy, but sulfur sublimates and redistributes in the high-vacuum conditions of conventional electron microscopes. The resulting sublimation artifacts render characterization of sulfur in conventional electron microscopes problematic and unreliable. Here, we demonstratemore » two techniques, cryogenic transmission electron microscopy (cryo-TEM) and scanning electron microscopy in air (airSEM), that enable the reliable characterization of sulfur across multiple length scales by suppressing sulfur sublimation. We use cryo-TEM and airSEM to examine carbon–sulfur composites synthesized for use as Li–S battery cathodes, noting several cases where the commonly employed sulfur melt infusion method is highly inefficient at infiltrating sulfur into porous carbon hosts.« less

Catalytic chemical vapor deposition synthesis and electron microscopy observation of coiled carbon nanotubes

NASA Astrophysics Data System (ADS)

Xie, Jining; Mukhopadyay, K.; Yadev, J.; Varadan, V. K.

2003-10-01

Coiled carbon nanotubes exhibit excellent mechanical and electrical properties because of the combination of coil morphology and properties of nanotubes. They could have potential novel applications in nanocomposites and nano-electronic devices as well as nano-electromechanical systems. In this work, synthesis of regularly coiled carbon nanotubes is presented. It involves pyrolysis of hydrocarbon gas over metal/support catalyst by both thermal filament and microwave catalytic chemical vapor deposition methods. Scanning electron microscopy and transmission electron microscopy were performed to observe the coil morphology and nanostructure of coiled nanotubes. The growth mechanism and structural and electrical properties of coiled carbon nanotubes are also discussed.

Possibilities and limitations of advanced transmission electron microscopy for carbon-based nanomaterials

PubMed Central

Bittencourt, Carla; Van Tendeloo, Gustaaf

2015-01-01

Summary A major revolution for electron microscopy in the past decade is the introduction of aberration correction, which enables one to increase both the spatial resolution and the energy resolution to the optical limit. Aberration correction has contributed significantly to the imaging at low operating voltages. This is crucial for carbon-based nanomaterials which are sensitive to electron irradiation. The research of carbon nanomaterials and nanohybrids, in particular the fundamental understanding of defects and interfaces, can now be carried out in unprecedented detail by aberration-corrected transmission electron microscopy (AC-TEM). This review discusses new possibilities and limits of AC-TEM at low voltage, including the structural imaging at atomic resolution, in three dimensions and spectroscopic investigation of chemistry and bonding. In situ TEM of carbon-based nanomaterials is discussed and illustrated through recent reports with particular emphasis on the underlying physics of interactions between electrons and carbon atoms. PMID:26425406

Evaluation of anterior lenticonus in alport syndrome using tracey wavefront aberrometry and transmission electron microscopy.

PubMed

Kim, Kwan Soo; Kim, Mo Sae; Kim, Joon Mo; Choi, Chul Young

2010-01-01

To evaluate the efficacy of Tracey wavefront aberrometry (Tracey Technologies, Houston, TX) and transmission electron microscopy for the detection of anterior lenticonus in Alport syndrome. Tracey wavefront aberrometry was used to treat a patient with bilateral anterior lenticonus who had a history of Alport syndrome. For transmission electron microscopic examination, anterior lens capsules were obtained during clear lens phacoemulsification and intraocular lens implantation. Spherical aberrations were the predominant higher-order aberrations in the internal optics of both eyes. The Tracey wavefront aberrometer showed that most of the irregular astigmatism originated from the lenticular portion. Transmission electron microscopy of the specimens showed anterior lens capsules with decreased thickness and multiple dehiscences. Tracey wavefront aberrometry and transmission electron microscopy are effective tools for evaluation of anterior lenticonus in Alport syndrome. Copyright 2010, SLACK Incorporated.

High-resolution structure of the Shigella type-III secretion needle by solid-state NMR and cryo-electron microscopy

NASA Astrophysics Data System (ADS)

Demers, Jean-Philippe; Habenstein, Birgit; Loquet, Antoine; Kumar Vasa, Suresh; Giller, Karin; Becker, Stefan; Baker, David; Lange, Adam; Sgourakis, Nikolaos G.

2014-09-01

We introduce a general hybrid approach for determining the structures of supramolecular assemblies. Cryo-electron microscopy (cryo-EM) data define the overall envelope of the assembly and rigid-body orientation of the subunits while solid-state nuclear magnetic resonance (ssNMR) chemical shifts and distance constraints define the local secondary structure, protein fold and inter-subunit interactions. Finally, Rosetta structure calculations provide a general framework to integrate the different sources of structural information. Combining a 7.7-Å cryo-EM density map and 996 ssNMR distance constraints, the structure of the type-III secretion system needle of Shigella flexneri is determined to a precision of 0.4 Å. The calculated structures are cross-validated using an independent data set of 691 ssNMR constraints and scanning transmission electron microscopy measurements. The hybrid model resolves the conformation of the non-conserved N terminus, which occupies a protrusion in the cryo-EM density, and reveals conserved pore residues forming a continuous pattern of electrostatic interactions, thereby suggesting a mechanism for effector protein translocation.

Size and Shape of Protein Molecules at the Nanometer Level Determined by Sedimentation, Gel Filtration, and Electron Microscopy

PubMed Central

2009-01-01

An important part of characterizing any protein molecule is to determine its size and shape. Sedimentation and gel filtration are hydrodynamic techniques that can be used for this medium resolution structural analysis. This review collects a number of simple calculations that are useful for thinking about protein structure at the nanometer level. Readers are reminded that the Perrin equation is generally not a valid approach to determine the shape of proteins. Instead, a simple guideline is presented, based on the measured sedimentation coefficient and a calculated maximum S, to estimate if a protein is globular or elongated. It is recalled that a gel filtration column fractionates proteins on the basis of their Stokes radius, not molecular weight. The molecular weight can be determined by combining gradient sedimentation and gel filtration, techniques available in most biochemistry laboratories, as originally proposed by Siegel and Monte. Finally, rotary shadowing and negative stain electron microscopy are powerful techniques for resolving the size and shape of single protein molecules and complexes at the nanometer level. A combination of hydrodynamics and electron microscopy is especially powerful. PMID:19495910

Active control of bright electron beams with RF optics for femtosecond microscopy

DOE PAGES

Williams, J.; Zhou, F.; Sun, T.; ...

2017-08-01

A frontier challenge in implementing femtosecond electron microscopy is to gain precise optical control of intense beams to mitigate collective space charge effects for significantly improving the throughput. In this paper, we explore the flexible uses of an RF cavity as a longitudinal lens in a high-intensity beam column for condensing the electron beams both temporally and spectrally, relevant to the design of ultrafast electron microscopy. Through the introduction of a novel atomic grating approach for characterization of electron bunch phase space and control optics, we elucidate the principles for predicting and controlling the phase space dynamics to reach optimalmore » compressions at various electron densities and generating conditions. We provide strategies to identify high-brightness modes, achieving ~100 fs and ~1 eV resolutions with 10 6 electrons per bunch, and establish the scaling of performance for different bunch charges. These results benchmark the sensitivity and resolution from the fundamental beam brightness perspective and also validate the adaptive optics concept to enable delicate control of the density-dependent phase space structures to optimize the performance, including delivering ultrashort, monochromatic, high-dose, or coherent electron bunches.« less

Active control of bright electron beams with RF optics for femtosecond microscopy

PubMed Central

Williams, J.; Zhou, F.; Sun, T.; Tao, Z.; Chang, K.; Makino, K.; Berz, M.; Duxbury, P. M.; Ruan, C.-Y.

2017-01-01

A frontier challenge in implementing femtosecond electron microscopy is to gain precise optical control of intense beams to mitigate collective space charge effects for significantly improving the throughput. Here, we explore the flexible uses of an RF cavity as a longitudinal lens in a high-intensity beam column for condensing the electron beams both temporally and spectrally, relevant to the design of ultrafast electron microscopy. Through the introduction of a novel atomic grating approach for characterization of electron bunch phase space and control optics, we elucidate the principles for predicting and controlling the phase space dynamics to reach optimal compressions at various electron densities and generating conditions. We provide strategies to identify high-brightness modes, achieving ∼100 fs and ∼1 eV resolutions with 106 electrons per bunch, and establish the scaling of performance for different bunch charges. These results benchmark the sensitivity and resolution from the fundamental beam brightness perspective and also validate the adaptive optics concept to enable delicate control of the density-dependent phase space structures to optimize the performance, including delivering ultrashort, monochromatic, high-dose, or coherent electron bunches. PMID:28868325

Liquid scanning transmission electron microscopy: imaging protein complexes in their native environment in whole eukaryotic cells.

PubMed

Peckys, Diana B; de Jonge, Niels

2014-04-01

Scanning transmission electron microscopy (STEM) of specimens in liquid, so-called Liquid STEM, is capable of imaging the individual subunits of macromolecular complexes in whole eukaryotic cells in liquid. This paper discusses this new microscopy modality within the context of state-of-the-art microscopy of cells. The principle of operation and equations for the resolution are described. The obtained images are different from those acquired with standard transmission electron microscopy showing the cellular ultrastructure. Instead, contrast is obtained on specific labels. Images can be recorded in two ways, either via STEM at 200 keV electron beam energy using a microfluidic chamber enclosing the cells, or via environmental scanning electron microscopy at 30 keV of cells in a wet environment. The first series of experiments involved the epidermal growth factor receptor labeled with gold nanoparticles. The labels were imaged in whole fixed cells with nanometer resolution. Since the cells can be kept alive in the microfluidic chamber, it is also feasible to detect the labels in unfixed, live cells. The rapid sample preparation and imaging allows studies of multiple whole cells.

A short story of imaging and spectroscopy of two-dimensional materials by scanning transmission electron microscopy.

PubMed

Idrobo, Juan C; Zhou, Wu

2017-09-01

Here we present a short historical account of when single adatom impurities where first identified in two-dimensional materials by scanning transmission electron microscopy (STEM). We also present a study of the graphene low-loss (below 50eV) response as a function of number of layers using electron energy-loss spectroscopy (EELS). The study shows that as few as three layers of graphene behave as bulk graphite for losses above 10eV We also show examples of how point and extended defects can easily be resolved and structural dynamics can be readily capture by using aberration-corrected STEM imaging. Finally, we show that the new generation of monochromators has opened up possibilities to explore new physics with an electron microscope. All these capabilities were enabled by the development of spherical aberration correctors and monochromators, where Ondrej Krivanek has played a key role. Copyright © 2017. Published by Elsevier B.V.

Scanning electron microscopy study of adhesion in sea urchin blastulae. M.S. Thesis

NASA Technical Reports Server (NTRS)

Crowther, Susan D.

1988-01-01

The dissociation supernatant (DS) isolated by disaggregating Strongylocentrotus purpuratus blastulae in calcium- and magnesium-free seawater specifically promotes reaggregation of S. purpuratus blastula cells. The purpose of this study was to use scanning electron microscopy to examine the gross morphology of aggregates formed in the presence of DS to see if it resembles adhesion in partially dissociated blastulae. A new reaggregation procedure developed here, using large volumes of cell suspension and a large diameter of rotation, was utilized to obtain sufficient quantities of aggregates for scanning electron microscopy. The results indicate that aggregates formed in the presence of DS resemble partially dissociated intact embryos in terms of the direct cell-cell adhesion observed. DS did not cause aggregation to form as a result of the entrapment of cells in masses of extracellular material. These studies provide the groundwork for further studies using transmission electron microscopy to more precisely define the adhesive contacts made by cells in the presence of the putative adhesion molecules present in DS.

Joint denoising and distortion correction of atomic scale scanning transmission electron microscopy images

NASA Astrophysics Data System (ADS)

Berkels, Benjamin; Wirth, Benedikt

2017-09-01

Nowadays, modern electron microscopes deliver images at atomic scale. The precise atomic structure encodes information about material properties. Thus, an important ingredient in the image analysis is to locate the centers of the atoms shown in micrographs as precisely as possible. Here, we consider scanning transmission electron microscopy (STEM), which acquires data in a rastering pattern, pixel by pixel. Due to this rastering combined with the magnification to atomic scale, movements of the specimen even at the nanometer scale lead to random image distortions that make precise atom localization difficult. Given a series of STEM images, we derive a Bayesian method that jointly estimates the distortion in each image and reconstructs the underlying atomic grid of the material by fitting the atom bumps with suitable bump functions. The resulting highly non-convex minimization problems are solved numerically with a trust region approach. Existence of minimizers and the model behavior for faster and faster rastering are investigated using variational techniques. The performance of the method is finally evaluated on both synthetic and real experimental data.

EMAN2: an extensible image processing suite for electron microscopy.

PubMed

Tang, Guang; Peng, Liwei; Baldwin, Philip R; Mann, Deepinder S; Jiang, Wen; Rees, Ian; Ludtke, Steven J

2007-01-01

EMAN is a scientific image processing package with a particular focus on single particle reconstruction from transmission electron microscopy (TEM) images. It was first released in 1999, and new versions have been released typically 2-3 times each year since that time. EMAN2 has been under development for the last two years, with a completely refactored image processing library, and a wide range of features to make it much more flexible and extensible than EMAN1. The user-level programs are better documented, more straightforward to use, and written in the Python scripting language, so advanced users can modify the programs' behavior without any recompilation. A completely rewritten 3D transformation class simplifies translation between Euler angle standards and symmetry conventions. The core C++ library has over 500 functions for image processing and associated tasks, and it is modular with introspection capabilities, so programmers can add new algorithms with minimal effort and programs can incorporate new capabilities automatically. Finally, a flexible new parallelism system has been designed to address the shortcomings in the rigid system in EMAN1.

Detection of parvoviruses in wolf feces by electron microscopy

USGS Publications Warehouse

Muneer, M.A.; Farah, I.O.; Pomeroy, K.A.; Goyal, S.M.; Mech, L.D.

1988-01-01

One hundred fifteen wolf (Canis lupus) feces were collected between 1980 and 1984 from northeastern Minnesota and were examined for canine parvovirus by negative contrast electron microscopy. Of these, seven (6%) samples revealed the presence of parvovirus. Some of these viruses were able to grow in cell cultures forming intranuclear inclusion bodies and giant cells.

A Global Approach for Quantitative Super Resolution and Electron Microscopy on Cryo and Epoxy Sections Using Self-labeling Protein Tags.

PubMed

Müller, Andreas; Neukam, Martin; Ivanova, Anna; Sönmez, Anke; Münster, Carla; Kretschmar, Susanne; Kalaidzidis, Yannis; Kurth, Thomas; Verbavatz, Jean-Marc; Solimena, Michele

2017-02-02

Correlative light and electron microscopy (CLEM) is a powerful approach to investigate the molecular ultrastructure of labeled cell compartments. However, quantitative CLEM studies are rare, mainly due to small sample sizes and the sensitivity of fluorescent proteins to strong fixatives and contrasting reagents for EM. Here, we show that fusion of a self-labeling protein to insulin allows for the quantification of age-distinct insulin granule pools in pancreatic beta cells by a combination of super resolution and transmission electron microscopy on Tokuyasu cryosections. In contrast to fluorescent proteins like GFP organic dyes covalently bound to self-labeling proteins retain their fluorescence also in epoxy resin following high pressure freezing and freeze substitution, or remarkably even after strong chemical fixation. This enables for the assessment of age-defined granule morphology and degradation. Finally, we demonstrate that this CLEM protocol is highly versatile, being suitable for single and dual fluorescent labeling and detection of different proteins with optimal ultrastructure preservation and contrast.

Accurate Virus Quantitation Using a Scanning Transmission Electron Microscopy (STEM) Detector in a Scanning Electron Microscope

DTIC Science & Technology

2017-06-29

Accurate Virus Quantitation Using a Scanning Transmission Electron Microscopy (STEM) Detector in a Scanning Electron Microscope Candace D Blancett1...L Norris2, Cynthia A Rossi4 , Pamela J Glass3, Mei G Sun1,* 1 Pathology Division, United States Army Medical Research Institute of Infectious...Diseases (USAMRIID), 1425 Porter Street, Fort Detrick, Maryland, 21702 2Biostatistics Division, United States Army Medical Research Institute of

Low-cost cryo-light microscopy stage fabrication for correlated light/electron microscopy.

PubMed

Carlson, David B; Evans, James E

2011-06-05

The coupling of cryo-light microscopy (cryo-LM) and cryo-electron microscopy (cryo-EM) poses a number of advantages for understanding cellular dynamics and ultrastructure. First, cells can be imaged in a near native environment for both techniques. Second, due to the vitrification process, samples are preserved by rapid physical immobilization rather than slow chemical fixation. Third, imaging the same sample with both cryo-LM and cryo-EM provides correlation of data from a single cell, rather than a comparison of "representative samples". While these benefits are well known from prior studies, the widespread use of correlative cryo-LM and cryo-EM remains limited due to the expense and complexity of buying or building a suitable cryogenic light microscopy stage. Here we demonstrate the assembly, and use of an inexpensive cryogenic stage that can be fabricated in any lab for less than $40 with parts found at local hardware and grocery stores. This cryo-LM stage is designed for use with reflected light microscopes that are fitted with long working distance air objectives. For correlative cryo-LM and cryo-EM studies, we adapt the use of carbon coated standard 3-mm cryo-EM grids as specimen supports. After adsorbing the sample to the grid, previously established protocols for vitrifying the sample and transferring/handling the grid are followed to permit multi-technique imaging. As a result, this setup allows any laboratory with a reflected light microscope to have access to direct correlative imaging of frozen hydrated samples.

Molecular tips for scanning tunneling microscopy: intermolecular electron tunneling for single-molecule recognition and electronics.

PubMed

Nishino, Tomoaki

2014-01-01

This paper reviews the development of molecular tips for scanning tunneling microscopy (STM). Molecular tips offer many advantages: first is their ability to perform chemically selective imaging because of chemical interactions between the sample and the molecular tip, thus improving a major drawback of conventional STM. Rational design of the molecular tip allows sophisticated chemical recognition; e.g., chiral recognition and selective visualization of atomic defects in carbon nanotubes. Another advantage is that they provide a unique method to quantify electron transfer between single molecules. Understanding such electron transfer is mandatory for the realization of molecular electronics.

Scanning electron microscopy analysis of corrosion degradation on tinplate substrates.

PubMed

Zumelzu, E; Cabezas, C; Vera, A

2003-01-01

The degradation of electrolytic tinplate used in food containers was analysed and evaluated, using scanning electron microscopy and electrochemical measurements of microcorrosion and ion dissolution by atomic absorption to prevent food contamination caused by metal traces and to increase the durability of such tinplates.

Scanning electron microscopy of echinoid podia.

PubMed

Florey, E; Cahill, M A

1982-01-01

Tube feet of the sea urchin Strongylocentrotus franciscanus were studied with the scanning electron microscope (SEM). By use of fractured preparations it was possible to obtain views of all components of the layered tube-foot wall. The outer epithelium was found to bear tufts of cilia possibly belonging to sensory cells. The nerve plexus was clearly revealed as being composed of bundles of varicose axons. The basal lamina, which covers the outer and inner surfaces of the connective tissue layer, was found to be a mechanically resistant and elastic membrane. The connective tissue appears as dense bundles of (collagen) fibers. The luminal epithelium (coelothelium) is a single layer of flagellated collar cells. There is no indication that the muscle fibers, which insert on the inner basal lamina of the connective tissue layer are innervated by axons from the basi-epithelial nerve plexus. The results agree with previous conclusions concerning tube-foot structure based on transmission electron microscopy, and provide additional information, particularly with regard to the outer and inner epithelia.

Preparation of gold nanocluster bioconjugates for electron microscopy.

PubMed

Heinecke, Christine L; Ackerson, Christopher J

2013-01-01

In this chapter, we describe types of gold nanoparticle-biomolecule conjugates and their use in electron microscopy. Included are two detailed protocols for labeling an IgG antibody with gold monolayer protected clusters. The first approach is a direct bonding approach that utilizes the ligand place exchange reaction. The second approach describes NHS-EDC coupling of Au(144)(pMBA)(60) with IgG. Also included are various characterization techniques for determining labeling efficiency.

A scanning electron microscopy study of the macro-crystalline structure of 2-(2,4-dinitrobenzyl) pyridine

NASA Technical Reports Server (NTRS)

Ware, Jacqueline; Hammond, Ernest C., Jr.

1989-01-01

The compound, 2-(2,4-dinitrobenzyl) pyridine, was synthesized in the laboratory; an introductory level electron microscopy study of the macro-crystalline structure was conducted using the scanning electron microscope (SEM). The structure of these crystals was compared with the macrostructure of the crystal of 2-(2,4-dinitrobenzyl) pyridinium bromide, the hydrobromic salt of the compound which was also synthesized in the laboratory. A scanning electron microscopy crystal study was combined with a study of the principle of the electron microscope.

Murphy's law-if anything can go wrong, it will: Problems in phage electron microscopy.

PubMed

Ackermann, Hans-W; Tiekotter, Kenneth L

2012-04-01

The quality of bacteriophage electron microscopy appears to be on a downward course since the 1980s. This coincides with the introduction of digital electron microscopes and a general lowering of standards, possibly due to the disappearance of several world-class electron microscopists The most important problem seems to be poor contrast. Positive staining is frequently not recognized as an undesirable artifact. Phage parts, bacterial debris, and aberrant or damaged phage particles may be misdiagnosed as bacterial viruses. Digital electron microscopes often seem to be operated without magnification control because this is difficult and inconvenient. In summary, most phage electron microscopy problems may be attributed to human failure. Journals are a last-ditch defense and have a heavy responsibility in selecting competent reviewers and rejecting, or not, unsatisfactory articles.

Functionalization of a nanostructured hydroxyapatite with Cu(II) compounds as a pesticide: in situ transmission electron microscopy and environmental scanning electron microscopy observations of treated Vitis vinifera L. leaves.

PubMed

Battiston, Enrico; Salvatici, Maria C; Lavacchi, Alessandro; Gatti, Antonietta; Di Marco, Stefano; Mugnai, Laura

2018-02-19

The present study evaluated a biocompatible material for plant protection with the aim of reducing the amount of active substance applied. We used a synthetic hydroxyapatite (HA) that has been studied extensively as a consequence of its bioactivity and biocompatibility. An aggregation between HA nanoparticles and four Cu(II) compounds applied to Vitis vinifera L. leaves as a pesticide was studied. Formulations were characterized by X-ray diffraction (XRD), dynamic light scattering (DLS) and electron microscopy and applied in planta to verify particle aggregation and efficiency in controlling the pathogen Plasmopara viticola. The XRD patterns showed different crystalline phases dependig on the Cu(II) compound formulated with HA particles, DLS showed that nanostructured particles are stable as aggregates out of the nanometer range and, in all formulations, transmission electron microscopy (TEM) and environmental scanning electron microscopy (ESEM) microscopy showed large aggregates which were partially nanostructured and were recognized as stable in their micrometric dimensions. Such particles did not show phytotoxic effects after their application in planta. A formulation based on HA and a soluble Cu(II) compound showed promising results in the control of the fungal pathogen, confirming the potential role of HA as an innovative delivery system of Cu(II) ions. The present work indicates the possibility of improving the biological activity of a bioactive substance by modifying its structure through an achievable formulation with a biocompatible material. © 2018 Society of Chemical Industry. © 2018 Society of Chemical Industry.

Fretting wear in titanium, Monel-400, and cobalt 25-percent-molybdenum using scanning electron microscopy

NASA Technical Reports Server (NTRS)

Bill, R. C.

1972-01-01

Damage scar volume measurements taken from like metal fretting pairs combined with scanning electron microscopy observations showed that three sequentially operating mechanisms result in the fretting of titanium, Monel-400, and cobalt - 25-percent molybdenum. Initially, adhesion and plastic deformation of the surface played an important role. This was followed after a few hundred cycles by a fatigue mechanism which produced spall-like pits in the damage scar. Finally, a combination of oxidation and abrasion by debris particles became most significant. Damage scar measurements made on several elemental metals after 600,000 fretting cycles suggested that the ratio of oxide hardness to metal hardness was a measure of the susceptibility of a metal to progressive damage by fretting.

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

PubMed Central

Hampton, Cheri M; Strauss, Joshua D; Ke, Zunlong; Dillard, Rebecca S; Hammonds, Jason E; Alonas, Eric; Desai, Tanay M; Marin, Mariana; Storms, Rachel E; Leon, Fredrick; Melikyan, Gregory B; Santangelo, Philip J; Spearman, Paul W; Wright, Elizabeth R

2016-01-01

Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5–15 d for an individual experienced in cryo-EM. PMID:27977021

Water without windows: Evaluating the performance of open cell transmission electron microscopy under saturated water vapor conditions, and assessing its potential for microscopy of hydrated biological specimens.

PubMed

Cassidy, Cathal; Yamashita, Masao; Cheung, Martin; Kalale, Chola; Adaniya, Hidehito; Kuwahara, Ryusuke; Shintake, Tsumoru

2017-01-01

We have performed open cell transmission electron microscopy experiments through pure water vapor in the saturation pressure regime (>0.6 kPa), in a modern microscope capable of sub-Å resolution. We have systematically studied achievable pressure levels, stability and gas purity, effective thickness of the water vapor column and associated electron scattering processes, and the effect of gas pressure on electron optical resolution and image contrast. For example, for 1.3 kPa pure water vapor and 300kV electrons, we report pressure stability of ± 20 Pa over tens of minutes, effective thickness of 0.57 inelastic mean free paths, lattice resolution of 0.14 nm on a reference Au specimen, and no significant degradation in contrast or stability of a biological specimen (M13 virus, with 6 nm body diameter). We have also done some brief experiments to confirm feasibility of loading specimens into an in situ water vapor ambient without exposure to intermediate desiccating conditions. Finally, we have also checked if water experiments had any discernible impact on the microscope performance, and report pertinent vacuum and electron optical data, for reference purposes.

Water without windows: Evaluating the performance of open cell transmission electron microscopy under saturated water vapor conditions, and assessing its potential for microscopy of hydrated biological specimens

PubMed Central

Yamashita, Masao; Cheung, Martin; Kalale, Chola; Adaniya, Hidehito; Kuwahara, Ryusuke; Shintake, Tsumoru

2017-01-01

We have performed open cell transmission electron microscopy experiments through pure water vapor in the saturation pressure regime (>0.6 kPa), in a modern microscope capable of sub-Å resolution. We have systematically studied achievable pressure levels, stability and gas purity, effective thickness of the water vapor column and associated electron scattering processes, and the effect of gas pressure on electron optical resolution and image contrast. For example, for 1.3 kPa pure water vapor and 300kV electrons, we report pressure stability of ± 20 Pa over tens of minutes, effective thickness of 0.57 inelastic mean free paths, lattice resolution of 0.14 nm on a reference Au specimen, and no significant degradation in contrast or stability of a biological specimen (M13 virus, with 6 nm body diameter). We have also done some brief experiments to confirm feasibility of loading specimens into an in situ water vapor ambient without exposure to intermediate desiccating conditions. Finally, we have also checked if water experiments had any discernible impact on the microscope performance, and report pertinent vacuum and electron optical data, for reference purposes. PMID:29099843

Discrete Chromatic Aberrations Arising from Photoinduced Electron-Photon Interactions in Ultrafast Electron Microscopy.

PubMed

Plemmons, Dayne A; Flannigan, David J

2016-05-26

In femtosecond ultrafast electron microscopy (UEM) experiments, the initial excitation period is composed of spatiotemporal overlap of the temporally commensurate pump photon pulse and probe photoelectron packet. Generation of evanescent near-fields at the nanostructure specimens produces a dispersion relation that enables coupling of the photons (ℏω = 2.4 eV, for example) and freely propagating electrons (200 keV, for example) in the near-field. Typically, this manifests as discrete peaks occurring at integer multiples (n) of the photon energy in the low-loss/gain region of electron-energy spectra (i.e., at 200 keV ± nℏω eV). Here, we examine the UEM imaging resolution implications of the strong inelastic near-field interactions between the photons employed in optical excitation and the probe photoelectrons. We find that the additional photoinduced energy dispersion occurring when swift electrons pass through intense evanescent near-fields results in a discrete chromatic aberration that limits the spatial resolving power to several angstroms during the excitation period.

Scanning electron microscopy imaging of dislocations in bulk materials, using electron channeling contrast.

PubMed

Crimp, Martin A

2006-05-01

The imaging and characterization of dislocations is commonly carried out by thin foil transmission electron microscopy (TEM) using diffraction contrast imaging. However, the thin foil approach is limited by difficult sample preparation, thin foil artifacts, relatively small viewable areas, and constraints on carrying out in situ studies. Electron channeling imaging of electron channeling contrast imaging (ECCI) offers an alternative approach for imaging crystalline defects, including dislocations. Because ECCI is carried out with field emission gun scanning electron microscope (FEG-SEM) using bulk specimens, many of the limitations of TEM thin foil analysis are overcome. This paper outlines the development of electron channeling patterns and channeling imaging to the current state of the art. The experimental parameters and set up necessary to carry out routine channeling imaging are reviewed. A number of examples that illustrate some of the advantages of ECCI over thin foil TEM are presented along with a discussion of some of the limitations on carrying out channeling contrast analysis of defect structures. Copyright (c) 2006 Wiley-Liss, Inc.

Fast imaging with inelastically scattered electrons by off-axis chromatic confocal electron microscopy.

PubMed

Zheng, Changlin; Zhu, Ye; Lazar, Sorin; Etheridge, Joanne

2014-04-25

We introduce off-axis chromatic scanning confocal electron microscopy, a technique for fast mapping of inelastically scattered electrons in a scanning transmission electron microscope without a spectrometer. The off-axis confocal mode enables the inelastically scattered electrons to be chromatically dispersed both parallel and perpendicular to the optic axis. This enables electrons with different energy losses to be separated and detected in the image plane, enabling efficient energy filtering in a confocal mode with an integrating detector. We describe the experimental configuration and demonstrate the method with nanoscale core-loss chemical mapping of silver (M4,5) in an aluminium-silver alloy and atomic scale imaging of the low intensity core-loss La (M4,5@840  eV) signal in LaB6. Scan rates up to 2 orders of magnitude faster than conventional methods were used, enabling a corresponding reduction in radiation dose and increase in the field of view. If coupled with the enhanced depth and lateral resolution of the incoherent confocal configuration, this offers an approach for nanoscale three-dimensional chemical mapping.

Electron-beam broadening in amorphous carbon films in low-energy scanning transmission electron microscopy.

PubMed

Drees, H; Müller, E; Dries, M; Gerthsen, D

2018-02-01

Resolution in scanning transmission electron microscopy (STEM) is ultimately limited by the diameter of the electron beam. The electron beam diameter is not only determined by the properties of the condenser lens system but also by electron scattering in the specimen which leads to electron-beam broadening and degradation of the resolution with increasing specimen thickness. In this work we introduce a new method to measure electron-beam broadening which is based on STEM imaging with a multi-segmented STEM detector. We focus on STEM at low electron energies between 10 and 30 keV and use an amorphous carbon film with known thickness as test object. The experimental results are compared with calculated beam diameters using different analytical models and Monte-Carlo simulations. We find excellent agreement of the experimental data with the recently published model by Gauvin and Rudinsky [1] for small t/λ el (thickness to elastic mean free path) values which are considered in our study. Copyright © 2017 Elsevier B.V. All rights reserved.

Nanodiamonds as multi-purpose labels for microscopy.

PubMed

Hemelaar, S R; de Boer, P; Chipaux, M; Zuidema, W; Hamoh, T; Martinez, F Perona; Nagl, A; Hoogenboom, J P; Giepmans, B N G; Schirhagl, R

2017-04-07

Nanodiamonds containing fluorescent nitrogen-vacancy centers are increasingly attracting interest for use as a probe in biological microscopy. This interest stems from (i) strong resistance to photobleaching allowing prolonged fluorescence observation times; (ii) the possibility to excite fluorescence using a focused electron beam (cathodoluminescence; CL) for high-resolution localization; and (iii) the potential use for nanoscale sensing. For all these schemes, the development of versatile molecular labeling using relatively small diamonds is essential. Here, we show the direct targeting of a biological molecule with nanodiamonds as small as 70 nm using a streptavidin conjugation and standard antibody labelling approach. We also show internalization of 40 nm sized nanodiamonds. The fluorescence from the nanodiamonds survives osmium-fixation and plastic embedding making them suited for correlative light and electron microscopy. We show that CL can be observed from epon-embedded nanodiamonds, while surface-exposed nanoparticles also stand out in secondary electron (SE) signal due to the exceptionally high diamond SE yield. Finally, we demonstrate the magnetic read-out using fluorescence from diamonds prior to embedding. Thus, our results firmly establish nanodiamonds containing nitrogen-vacancy centers as unique, versatile probes for combining and correlating different types of microscopy, from fluorescence imaging and magnetometry to ultrastructural investigation using electron microscopy.

Electron microscopy characterization of AlGaN/GaN heterostructures grown on Si (111) substrates

NASA Astrophysics Data System (ADS)

Gkanatsiou, A.; Lioutas, Ch. B.; Frangis, N.; Polychroniadis, E. K.; Prystawko, P.; Leszczynski, M.

2017-03-01

AlGaN/GaN buffer heterostructures were grown on "on axis" and 4 deg off Si (111) substrates by MOVPE. The electron microscopy study reveals the very good epitaxial growth of the layers. Almost c-plane orientated nucleation grains are achieved after full AlN layer growth. Step-graded AlGaN layers were introduced, in order to prevent the stress relaxation and to work as a dislocation filter. Thus, a crack-free smooth surface of the final GaN epitaxial layer is achieved in both cases, making the buffer structure ideal for the forthcoming growth of the heterostructure (used for HEMT device applications). Finally, the growth of the AlGaN/GaN heterostructure on top presents characteristic and periodic undulations (V-pits) on the surface, due to strain relaxation reasons. The AlN interlayer grown in between the heterostructure demonstrates an almost homogeneous thickness, probably reinforcing the 2DEG electrical characteristics.

Weak-beam scanning transmission electron microscopy for quantitative dislocation density measurement in steels.

PubMed

Yoshida, Kenta; Shimodaira, Masaki; Toyama, Takeshi; Shimizu, Yasuo; Inoue, Koji; Yoshiie, Toshimasa; Milan, Konstantinovic J; Gerard, Robert; Nagai, Yasuyoshi

2017-04-01

To evaluate dislocations induced by neutron irradiation, we developed a weak-beam scanning transmission electron microscopy (WB-STEM) system by installing a novel beam selector, an annular detector, a high-speed CCD camera and an imaging filter in the camera chamber of a spherical aberration-corrected transmission electron microscope. The capabilities of the WB-STEM with respect to wide-view imaging, real-time diffraction monitoring and multi-contrast imaging are demonstrated using typical reactor pressure vessel steel that had been used in an European nuclear reactor for 30 years as a surveillance test piece with a fluence of 1.09 × 1020 neutrons cm-2. The quantitatively measured size distribution (average loop size = 3.6 ± 2.1 nm), number density of the dislocation loops (3.6 × 1022 m-3) and dislocation density (7.8 × 1013 m m-3) were carefully compared with the values obtained via conventional weak-beam transmission electron microscopy studies. In addition, cluster analysis using atom probe tomography (APT) further demonstrated the potential of the WB-STEM for correlative electron tomography/APT experiments. © The Author 2017. Published by Oxford University Press on behalf of The Japanese Society of Microscopy. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

A compilation of cold cases using scanning electron microscopy at the University of Rhode Island

NASA Astrophysics Data System (ADS)

Platek, Michael J.; Gregory, Otto J.

2015-10-01

Scanning electron microscopy combined with microchemical analysis has evolved into one of the most widely used instruments in forensic science today. In particular, the environmental scanning electron microscope (SEM) in conjunction with energy dispersive spectroscopy (EDS), has created unique opportunities in forensic science in regard to the examination of trace evidence; i.e. the examination of evidence without altering the evidence with conductive coatings, thereby enabling criminalists to solve cases that were previously considered unsolvable. Two cold cases were solved at URI using a JEOL 5900 LV SEM in conjunction with EDS. A cold case murder and a cold missing person case will be presented from the viewpoint of the microscopist and will include sample preparation, as well as image and chemical analysis of the trace evidence using electron microscopy and optical microscopy.

Electron Microscopy of Staphylococcus aureus Cell Wall Lysis

PubMed Central

Virgilio, R.; González, C.; Muñoz, Nubia; Mendoza, Silvia

1966-01-01

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Muñoz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018–2024. 1966.—A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents. Images PMID:5939482

Electron microscopy of Staphylococcus aureus cell wall lysis.

PubMed

Virgilio, R; González, C; Muñoz, N; Mendoza, S

1966-05-01

Virgilio, Rafael (Escuela de Química y Farmacia, Universidad de Chile, Santiago, Chile), C. González, Nubia Muñoz, and Silvia Mendoza. Electron microscopy of Staphylococcus aureus cell wall lysis. J. Bacteriol. 91:2018-2024. 1966.-A crude suspension of Staphylococcus aureus cell walls (strain Cowan III) in buffer solution was shown by electron microscopy to lyse slightly after 16 hr, probably owing to the action of autolysin. The lysis was considerably faster and more intense after the addition of lysozyme. A remarkable reduction in thickness and rigidity of the cell walls, together with the appearance of many irregular protrusions in their outlines, was observed after 2 hr; after 16 hr, there remained only a few recognizable cell wall fragments but many residual particulate remnants. When autolysin was previously inactivated by trypsin, there was a complete inhibition of the lytic action of lysozyme; on the other hand, when autolysin was inactivated by heat and lysozyme was added, a distinct decrease in the thickness of the cell walls was observed, but there was no destruction of the walls. The lytic action of lysozyme, after treatment with hot 5% trichloroacetic acid, gave rise to a marked dissolution of the structure of the cell walls, which became lost against the background, without, however, showing ostensible alteration of wall outlines. From a morphological point of view, the lytic action of autolysin plus lysozyme was quite different from that of trichloroacetic acid plus lysozyme, as shown by electron micrographs, but in both cases it was very intense. This would suggest different mechanisms of action for these agents.

Challenges of microtome‐based serial block‐face scanning electron microscopy in neuroscience

PubMed Central

WANNER, A. A.; KIRSCHMANN, M. A.

2015-01-01

Summary Serial block‐face scanning electron microscopy (SBEM) is becoming increasingly popular for a wide range of applications in many disciplines from biology to material sciences. This review focuses on applications for circuit reconstruction in neuroscience, which is one of the major driving forces advancing SBEM. Neuronal circuit reconstruction poses exceptional challenges to volume EM in terms of resolution, field of view, acquisition time and sample preparation. Mapping the connections between neurons in the brain is crucial for understanding information flow and information processing in the brain. However, information on the connectivity between hundreds or even thousands of neurons densely packed in neuronal microcircuits is still largely missing. Volume EM techniques such as serial section TEM, automated tape‐collecting ultramicrotome, focused ion‐beam scanning electron microscopy and SBEM (microtome serial block‐face scanning electron microscopy) are the techniques that provide sufficient resolution to resolve ultrastructural details such as synapses and provides sufficient field of view for dense reconstruction of neuronal circuits. While volume EM techniques are advancing, they are generating large data sets on the terabyte scale that require new image processing workflows and analysis tools. In this review, we present the recent advances in SBEM for circuit reconstruction in neuroscience and an overview of existing image processing and analysis pipelines. PMID:25907464

Morphology of the ferritin iron core by aberration corrected scanning transmission electron microscopy

NASA Astrophysics Data System (ADS)

Jian, Nan; Dowle, Miriam; Horniblow, Richard D.; Tselepis, Chris; Palmer, Richard E.

2016-11-01

As the major iron storage protein, ferritin stores and releases iron for maintaining the balance of iron in fauna, flora, and bacteria. We present an investigation of the morphology and iron loading of ferritin (from equine spleen) using aberration-corrected high angle annular dark field scanning transmission electron microscopy. Atom counting method, with size selected Au clusters as mass standards, was employed to determine the number of iron atoms in the nanoparticle core of each ferritin protein. Quantitative analysis shows that the nuclearity of iron atoms in the mineral core varies from a few hundred iron atoms to around 5000 atoms. Moreover, a relationship between the iron loading and iron core morphology is established, in which mineral core nucleates from a single nanoparticle, then grows along the protein shell before finally forming either a solid or hollow core structure.

High-pressure freezing and freeze substitution of Arabidopsis for electron microscopy.

PubMed

Austin, Jotham R

2014-01-01

The objectives of electron microscopy ultrastructural studies are to examine cellular architecture and relate the cell's structural machinery to dynamic functional roles. This aspiration is difficult to achieve if specimens have not been adequately preserved in a "living state"; hence specimen preparation is of the utmost importance for the success of any electron micrographic study. High-pressure freezing (HPF)/freeze substitution (FS) has long been recognized as the primer technique for the preservation of ultrastructure in biological samples. In most cases a basic HPF/freeze substitution protocol is sufficient to obtain superior ultrastructural preservation and structural contrast, which allows one to use more advanced microscopy techniques such as 3D electron tomography. However, for plant tissues, which have a thick cell wall, large water-filled vacuoles, and air spaces (all of which are detrimental to cryopreservation), these basic HPF/FS protocols often yield undesirable results. In particular, ice crystal artifacts and the staining of membrane systems are often poorly or negatively stained, which make 3D segmentation of a tomogram difficult. To overcome these problems, various aspects of the HPF/FS protocol can be altered, including the cryo-filler(s) used, freeze substitution cocktail, and the resin infiltration process. This chapter will describe these modifications for the preparation of plant tissues for routine electron microscopic studies, immunocytochemistry, and 3D tomographic electron imaging.

Quantitative light and scanning electron microscopy of ferret sperm.

PubMed

Van der Horst, G; Curry, P T; Kitchin, R M; Burgess, W; Thorne, E T; Kwiatkowski, D; Parker, M; Atherton, R W

1991-11-01

Sperm were obtained via electroejaculation from Domestic ferret, (Mustela putorius furo), Siberian ferret (M. eversmanni), Black-footed ferret (M. nigripes), and a hybrid between Siberian and Domestic, called the Fitch ferret (M. sp.). Comparisons of sperm were made by four different microscopy techniques to determine whether differences exist among species. First, Nomarski differential interference microscopy could be used to distinguish domestic ferret sperm from the others on the basis of the structure of the posterior part of the acrosome. Second, both silver staining, which demonstrates argentophilic protein distribution, and scanning electron microscopy (SEM), revealed differences among the morphology of sperm for each species; variation in the unique appearance of the acrosome in ferret sperm was detected especially well by SEM. To quantify differences in morphology, five sperm head parameters were measured using image analysis; light microscopy produced significantly larger values than did SEM (all parameters and all species but Fitch), and there were significant differences owing to species for all parameters but one. Generally, our data demonstrate the value of complementary techniques to distinguish among sperm of closely related species and more specifically may help establish evolutionary relationships among the ferret species studied. In addition, they provide baseline data important for the captive breeding of the endangered Black-footed ferret.

Scanning electron microscopy of tinea nigra.

PubMed

Guarenti, Isabelle Maffei; Almeida, Hiram Larangeira de; Leitão, Aline Hatzenberger; Rocha, Nara Moreira; Silva, Ricardo Marques E

2014-01-01

Tinea nigra is a rare superficial mycosis caused by Hortaea werneckii. This infection presents as asymptomatic brown to black maculae mostly in palmo-plantar regions. We performed scanning electron microscopy of a superficial shaving of a tinea nigra lesion. The examination of the outer surface of the sample showed the epidermis with corneocytes and hyphae and elimination of fungal filaments. The inner surface of the sample showed important aggregation of hyphae among keratinocytes, which formed small fungal colonies. The ultrastructural findings correlated with those of dermoscopic examination - the small fungal aggregations may be the dark spicules seen on dermoscopy - and also allowed to document the mode of dissemination of tinea nigra, showing how hyphae are eliminated on the surface of the lesion.

Scanning electron microscopy of tinea nigra*

PubMed Central

Guarenti, Isabelle Maffei; de Almeida, Hiram Larangeira; Leitão, Aline Hatzenberger; Rocha, Nara Moreira; Silva, Ricardo Marques e

2014-01-01

Tinea nigra is a rare superficial mycosis caused by Hortaea werneckii. This infection presents as asymptomatic brown to black maculae mostly in palmo-plantar regions. We performed scanning electron microscopy of a superficial shaving of a tinea nigra lesion. The examination of the outer surface of the sample showed the epidermis with corneocytes and hyphae and elimination of fungal filaments. The inner surface of the sample showed important aggregation of hyphae among keratinocytes, which formed small fungal colonies. The ultrastructural findings correlated with those of dermoscopic examination - the small fungal aggregations may be the dark spicules seen on dermoscopy - and also allowed to document the mode of dissemination of tinea nigra, showing how hyphae are eliminated on the surface of the lesion. PMID:24770516

Quantifying Nanoscale Order in Amorphous Materials via Fluctuation Electron Microscopy

ERIC Educational Resources Information Center

Bogle, Stephanie Nicole

2009-01-01

Fluctuation electron microscopy (FEM) has been used to study the nanoscale order in various amorphous materials. The method is explicitly sensitive to 3- and 4-body atomic correlation functions in amorphous materials; this is sufficient to establish the existence of structural order on the nanoscale, even when the radial distribution function…

Collaboration at the Nanoscale: Exploring Viral Genetics with Electron Microscopy

ERIC Educational Resources Information Center

Duboise, S. Monroe; Moulton, Karen D.; Jamison, Jennifer L.

2009-01-01

The Maine Science Corps is a project sponsored by the National Science Foundation's (NSF) Graduate Teaching Fellows in K-12 Education (GK-12 ) program. Through this program, the University of Southern Maine's (USM) virology and transmission electron microscopy (TEM) research group provides high school teachers and students in rural areas with…

Correlative scanning-transmission electron microscopy reveals that a chimeric flavivirus is released as individual particles in secretory vesicles.

PubMed

Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

2014-01-01

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations.

Correlative Scanning-Transmission Electron Microscopy Reveals that a Chimeric Flavivirus Is Released as Individual Particles in Secretory Vesicles

PubMed Central

Burlaud-Gaillard, Julien; Sellin, Caroline; Georgeault, Sonia; Uzbekov, Rustem; Lebos, Claude; Guillaume, Jean-Marc; Roingeard, Philippe

2014-01-01

The intracellular morphogenesis of flaviviruses has been well described, but flavivirus release from the host cell remains poorly documented. We took advantage of the optimized production of an attenuated chimeric yellow fever/dengue virus for vaccine purposes to study this phenomenon by microscopic approaches. Scanning electron microscopy (SEM) showed the release of numerous viral particles at the cell surface through a short-lived process. For transmission electron microscopy (TEM) studies of the intracellular ultrastructure of the small number of cells releasing viral particles at a given time, we developed a new correlative microscopy method: CSEMTEM (for correlative scanning electron microscopy - transmission electron microscopy). CSEMTEM analysis suggested that chimeric flavivirus particles were released as individual particles, in small exocytosis vesicles, via a regulated secretory pathway. Our morphological findings provide new insight into interactions between flaviviruses and cells and demonstrate that CSEMTEM is a useful new method, complementary to SEM observations of biological events by intracellular TEM investigations. PMID:24681578

Ultrafast core-loss spectroscopy in four-dimensional electron microscopy

PubMed Central

van der Veen, Renske M.; Penfold, Thomas J.; Zewail, Ahmed H.

2015-01-01

We demonstrate ultrafast core-electron energy-loss spectroscopy in four-dimensional electron microscopy as an element-specific probe of nanoscale dynamics. We apply it to the study of photoexcited graphite with femtosecond and nanosecond resolutions. The transient core-loss spectra, in combination with ab initio molecular dynamics simulations, reveal the elongation of the carbon-carbon bonds, even though the overall behavior is a contraction of the crystal lattice. A prompt energy-gap shrinkage is observed on the picosecond time scale, which is caused by local bond length elongation and the direct renormalization of band energies due to temperature-dependent electron–phonon interactions. PMID:26798793

The microscopic world: A demonstration of electron microscopy for younger students

NASA Technical Reports Server (NTRS)

Horton, Linda L.

1992-01-01

The purpose is to excite students about the importance of scientific investigation and demonstrate why they should look at things in greater detail, extending beyond superficial examination. The topics covered include: microscopy, scanning electron microscopes, high magnification, and the scientific method.

Ballistic Electron Emission Microscopy Studies of Ferromagnet - Semiconductor Interfaces

NASA Astrophysics Data System (ADS)

Mather, P. G.; Perrella, A. C.; Yurtsever, A.; Buhrman, R. A.

2004-03-01

Devices that employ spin as well as charge effects have been the subjects of extensive study recently. The magnetic tunneling transistor (1) is one important device that demonstrates an electrical means of injecting spin-polarized electrons into a semiconductor. A Schottky barrier lies at the heart of the device, and a high quality spatially homogenous and uniform barrier formed on GaAs is highly desirable. We have used ballistic electron emission microscopy (BEEM) to study CoFe, Fe and permalloy deposited on a GaAs substrate to give nanometer resolved evaluation of hot electron transport through the films and across the Schottky barrier. All films give a homogenous, uniform barrier as compared with evaporated Au/GaAs and Ag/GaAs interfaces. We will report on BEEM measurements of the hot electron transfer ratio across the Schottky barrier for the different ferromagnetic materials, and on the energy and spin-dependent hot electron attenuation lengths of the CoFe, Fe, and permalloy films. (1) Sebastiaan van Dijken, Xin Jiang, Stuart S. P. Parkin, APL, 80, 3364.

Four-dimensional ultrafast electron microscopy of phase transitions

PubMed Central

Grinolds, Michael S.; Lobastov, Vladimir A.; Weissenrieder, Jonas; Zewail, Ahmed H.

2006-01-01

Reported here is direct imaging (and diffraction) by using 4D ultrafast electron microscopy (UEM) with combined spatial and temporal resolutions. In the first phase of UEM, it was possible to obtain snapshot images by using timed, single-electron packets; each packet is free of space–charge effects. Here, we demonstrate the ability to obtain sequences of snapshots (“movies”) with atomic-scale spatial resolution and ultrashort temporal resolution. Specifically, it is shown that ultrafast metal–insulator phase transitions can be studied with these achieved spatial and temporal resolutions. The diffraction (atomic scale) and images (nanometer scale) we obtained manifest the structural phase transition with its characteristic hysteresis, and the time scale involved (100 fs) is now studied by directly monitoring coordinates of the atoms themselves. PMID:17130445

A Modular Hierarchical Approach to 3D Electron Microscopy Image Segmentation

PubMed Central

Liu, Ting; Jones, Cory; Seyedhosseini, Mojtaba; Tasdizen, Tolga

2014-01-01

The study of neural circuit reconstruction, i.e., connectomics, is a challenging problem in neuroscience. Automated and semi-automated electron microscopy (EM) image analysis can be tremendously helpful for connectomics research. In this paper, we propose a fully automatic approach for intra-section segmentation and inter-section reconstruction of neurons using EM images. A hierarchical merge tree structure is built to represent multiple region hypotheses and supervised classification techniques are used to evaluate their potentials, based on which we resolve the merge tree with consistency constraints to acquire final intra-section segmentation. Then, we use a supervised learning based linking procedure for the inter-section neuron reconstruction. Also, we develop a semi-automatic method that utilizes the intermediate outputs of our automatic algorithm and achieves intra-segmentation with minimal user intervention. The experimental results show that our automatic method can achieve close-to-human intra-segmentation accuracy and state-of-the-art inter-section reconstruction accuracy. We also show that our semi-automatic method can further improve the intra-segmentation accuracy. PMID:24491638

Using advanced electron microscopy for the characterization of catalytic materials

NASA Astrophysics Data System (ADS)

Pyrz, William D.

-corrected electron microscopy was used to systematically examine, atomic column by atomic column, the effect of elemental substitution on the long-range crystalline order, atomic coordinates, and site occupancies of the various formulations such that trends could be developed linking these properties to catalytic yields. To accomplish this task, an algorithm was developed that enabled the direct extraction of atomic coordinates and site occupancies from high-angle annular dark-field (HAADF) images to within 1% and 15% uncertainty, respectively. Furthermore, this general method could be applied to various crystalline systems and may dramatically improve the quality of initial structural models used in Rietveld refinements. Improvement in the quality of starting models may increase the structural and chemical complexity of inorganic structures that can be solved by using "powder methods" alone. In addition to the development of these trends, HAADF analyses also revealed the presence of coherent compositional miscibility gaps, rotational twin domains, and structural intergrowths in the complex Mo-V-M-O oxide system. Other catalytic systems that are addressed in this dissertation include Pd, Ag, and bimetallic Pd-Ag catalysts for the selective hydrogenation of acetylene in excess ethylene, alkali and alkaline earth promoted Ru catalysts for the production of clean hydrogen through the decomposition of ammonia, the production of Pt nanoparticles using dendrimer templates, and Pt-Re bimetallic catalysts for the conversion of glycerol to hydrocarbons and syn gas. In each of these studies, electron microscopy was used as a complimentary tool to synthetic and reaction studies to better understand interactions between the nanoparticles and the support/template, to determine the effect of adding various promoters, or to understand the nanoscale structural and chemical changes associated with the formation of bimetallic nanoparticles. A final area addressed in this dissertation is the

Advantages of indium-tin oxide-coated glass slides in correlative scanning electron microscopy applications of uncoated cultured cells.

PubMed

Pluk, H; Stokes, D J; Lich, B; Wieringa, B; Fransen, J

2009-03-01

A method of direct visualization by correlative scanning electron microscopy (SEM) and fluorescence light microscopy of cell structures of tissue cultured cells grown on conductive glass slides is described. We show that by growing cells on indium-tin oxide (ITO)-coated glass slides, secondary electron (SE) and backscatter electron (BSE) images of uncoated cells can be obtained in high-vacuum SEM without charging artefacts. Interestingly, we observed that BSE imaging is influenced by both accelerating voltage and ITO coating thickness. By combining SE and BSE imaging with fluorescence light microscopy imaging, we were able to reveal detailed features of actin cytoskeletal and mitochondrial structures in mouse embryonic fibroblasts. We propose that the application of ITO glass as a substrate for cell culture can easily be extended and offers new opportunities for correlative light and electron microscopy studies of adherently growing cells.

Gold nanoparticle uptake in whole cells in liquid examined by environmental scanning electron microscopy.

PubMed

Peckys, Diana B; de Jonge, Niels

2014-02-01

The size of gold nanoparticles (AuNPs) can influence various aspects of their cellular uptake. Light microscopy is not capable of resolving most AuNPs, while electron microscopy (EM) is not practically capable of acquiring the necessary statistical data from many cells and the results may suffer from various artifacts. Here, we demonstrate the use of a fast EM method for obtaining high-resolution data from a much larger population of cells than is usually feasible with conventional EM. A549 (human lung carcinoma) cells were subjected to uptake protocols with 10, 15, or 30 nm diameter AuNPs with adsorbed serum proteins. After 20 min, 24 h, or 45 h, the cells were fixed and imaged in whole in a thin layer of liquid water with environmental scanning electron microscopy equipped with a scanning transmission electron microscopy detector. The fast preparation and imaging of 145 whole cells in liquid allowed collection of nanoscale data within an exceptionally small amount of time of ~80 h. Analysis of 1,041 AuNP-filled vesicles showed that the long-term AuNP storing lysosomes increased their average size by 80 nm when AuNPs with 30 nm diameter were uptaken, compared to lysosomes of cells incubated with AuNPs of 10 and 15 nm diameter.

Ultrastructural analysis of testicular tissue and sperm by transmission and scanning electron microscopy.

PubMed

Chemes, Hector E

2013-01-01

Transmission electron microscopy (TEM) studies have provided the basis for an in-depth understanding of the cell biology and normal functioning of the testis and male gametes and have opened the way to characterize the functional role played by specific organelles in spermatogenesis and sperm function. The development of the scanning electron microscope (SEM) extended these boundaries to the recognition of cell and organ surface features and the architectural array of cells and tissues. The merging of immunocytochemical and histochemical approaches with electron microscopy has completed a series of technical improvements that integrate structural and functional features to provide a broad understanding of cell biology in health and disease. With these advances the detailed study of the intricate structural and molecular organization as well as the chemical composition of cellular organelles is now possible. Immunocytochemistry is used to identify proteins or other components and localize them in specific cells or organelles with high specificity and sensitivity, and histochemistry can be used to understand their function (i.e., enzyme activity). When these techniques are used in conjunction with electron microscopy their resolving power is further increased to subcellular levels. In the present chapter we will describe in detail various ultrastructural techniques that are now available for basic or translational research in reproductive biology and reproductive medicine. These include TEM, ultrastructural immunocytochemistry, ultrastructural histochemistry, and SEM.

Comparative morphology of zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussel sperm: Light and electron microscopy

USGS Publications Warehouse

Walker, G.K.; Black, M.G.; Edwards, C.A.

1996-01-01

Adult zebra (Dreissena polymorpha) and quagga (Dreissena bugensis) mussels were induced to release large quantities of live spermatozoa by the administration of 5-hydroxytryptamine (serotonin). Sperm were photographed alive using phase-contrast microscopy and were fixed subsequently with glutaraldehyde followed by osmium tetroxide for eventual examination by transmission or scanning electron microscopy. The sperm of both genera are of the ect-aquasperm type. Their overall dimensions and shape allow for easy discrimination at the light and scanning electron microscopy level. Transmission electron microscopy of the cells reveals a barrel-shaped nucleus in zebra mussel sperm and an elongated nucleus in quagga mussel sperm. In both species, an acrosome is cradled in a nuclear fossa. The ultrastructure of the acrosome and axial body, however, is distinctive for each species. The structures of the midpiece are shown, including a unique mitochondrial "skirt" that includes densely packed parallel cristae and extends in a narrow sheet from the mitochondria.

Tools for Model Building and Optimization into Near-Atomic Resolution Electron Cryo-Microscopy Density Maps.

PubMed

DiMaio, F; Chiu, W

2016-01-01

Electron cryo-microscopy (cryoEM) has advanced dramatically to become a viable tool for high-resolution structural biology research. The ultimate outcome of a cryoEM study is an atomic model of a macromolecule or its complex with interacting partners. This chapter describes a variety of algorithms and software to build a de novo model based on the cryoEM 3D density map, to optimize the model with the best stereochemistry restraints and finally to validate the model with proper protocols. The full process of atomic structure determination from a cryoEM map is described. The tools outlined in this chapter should prove extremely valuable in revealing atomic interactions guided by cryoEM data. © 2016 Elsevier Inc. All rights reserved.

Electron Microscopy Characterization of Vanadium Dioxide Thin Films and Nanoparticles

NASA Astrophysics Data System (ADS)

Rivera, Felipe

relationships accounts for the majority of the VO_2 grains observed, due to the sapphire substrate's geometry there were variations within these rules that changed the orientation of VO_2 grains with respect to the substrate's normal direction. 2) For the TiO_2, a substrate with a lower lattice mismatch, we observe the expected relationship where the rutile VO_2 [100], [110], and [001] crystal directions lie parallel to the TiO_2 substrate's [100], [110], and [001] crystal directions respectively. 3) For the amorphous SiO_2 layer, all VO_2 crystals that were measurable (those that grew to the thickness of the deposited film) had a preferred orientation with the the rutile VO_2[001] crystal direction tending to lie parallel to the plane of the specimen. The use of transmission electron microscopy (TEM) is presented as a tool for further characterization studies of this material and its applications. In this work TEM diffraction patterns taken from cross-sections of particles of the a- and r-cut sapphire substrates not only solidified the predominant family mentioned, but also helped lift the ambiguity present in the rutile VO_2{100} axes. Finally, a focused-ion beam technique for preparation of cross-sectional TEM samples of metallic thin films deposited on polymer substrates is demonstrated.

Studying localized corrosion using liquid cell transmission electron microscopy

DOE PAGES

Chee, See Wee; Pratt, Sarah H.; Hattar, Khalid; ...

2014-11-07

Using liquid cell transmission electron microscopy (LCTEM), localized corrosion of Cu and Al thin films immersed in aqueous NaCl solutions was studied. We demonstrate that potentiostatic control can be used to initiate pitting and that local compositional changes, due to focused ion beam implantation of Au + ions, can modify the corrosion susceptibility of Al films. Likewise, a discussion on strategies to control the onset of pitting is also presented.

Use of electron microscopy to classify canine perivascular wall tumors.

PubMed

Palmieri, C; Avallone, G; Cimini, M; Roccabianca, P; Stefanello, D; Della Salda, L

2013-03-01

The histologic classification of canine perivascular wall tumors (PWTs) is controversial. Many PWTs are still classified as hemangiopericytomas (HEPs), and the distinction from peripheral nerve sheath tumors (PNSTs) is still under debate. A recent histologic classification of canine soft tissue sarcomas included most histologic types of PWT but omitted those that were termed undifferentiated. Twelve cases of undifferentiated canine PWTs were evaluated by transmission electron microscopy. The ultrastructural findings supported a perivascular wall origin for all cases with 4 categories of differentiation: myopericytic (n = 4), myofibroblastic (n = 1), fibroblastic (n = 2), and undifferentiated (n = 5). A PNST was considered unlikely in each case based on immunohistochemical expression of desmin and/or the lack of typical ultrastructural features, such as basal lamina. Electron microscopy was pivotal for the subclassification of canine PWTs, and the results support the hypothesis that canine PWTs represent a continuum paralleling the phenotypic plasticity of vascular mural cells. The hypothesis that a subgroup of PWTs could arise from a pluripotent mesenchymal perivascular wall cell was also considered and may explain the diverse differentiation of canine PWTs.

Relative merits and limiting factors for x-ray and electron microscopy of thick, hydrated organic materials

DOE PAGES

Du, Ming; Jacobsen, Chris

2017-10-07

Electron and x-ray microscopes allow one to image the entire, unlabeled structure of hydrated materials at a resolution well beyond what visible light microscopes can achieve. However, both approaches involve ionizing radiation, so that radiation damage must be considered as one of the limits to imaging. Drawing upon earlier work, we describe here a unified approach to estimating the image contrast (and thus the required exposure and corresponding radiation dose) in both x-ray and electron microscopy. This approach accounts for factors such as plural and inelastic scattering, and (in electron microscopy) the use of energy filters to obtain so-called "zeromore » loss" images. As expected, it shows that electron microscopy offers lower dose for specimens thinner than about 1 mu m (such as for studies of macromolecules, viruses, bacteria and archaebacteria, and thin sectioned material), while x-ray microscopy offers superior characteristics for imaging thicker specimen such as whole eukaryotic cells, thick-sectioned tissues, and organs. The required radiation dose scales strongly as a function of the desired spatial resolution, allowing one to understand the limits of live and frozen hydrated specimen imaging. Lastly, we consider the factors limiting x-ray microscopy of thicker materials, suggesting that specimens as thick as a whole mouse brain can be imaged with x-ray microscopes without significant image degradation should appropriate image reconstruction methods be identified.« less

Relative merits and limiting factors for x-ray and electron microscopy of thick, hydrated organic materials

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Ming; Jacobsen, Chris

Electron and x-ray microscopes allow one to image the entire, unlabeled structure of hydrated materials at a resolution well beyond what visible light microscopes can achieve. However, both approaches involve ionizing radiation, so that radiation damage must be considered as one of the limits to imaging. Drawing upon earlier work, we describe here a unified approach to estimating the image contrast (and thus the required exposure and corresponding radiation dose) in both x-ray and electron microscopy. This approach accounts for factors such as plural and inelastic scattering, and (in electron microscopy) the use of energy filters to obtain so-called "zeromore » loss" images. As expected, it shows that electron microscopy offers lower dose for specimens thinner than about 1 mu m (such as for studies of macromolecules, viruses, bacteria and archaebacteria, and thin sectioned material), while x-ray microscopy offers superior characteristics for imaging thicker specimen such as whole eukaryotic cells, thick-sectioned tissues, and organs. The required radiation dose scales strongly as a function of the desired spatial resolution, allowing one to understand the limits of live and frozen hydrated specimen imaging. Lastly, we consider the factors limiting x-ray microscopy of thicker materials, suggesting that specimens as thick as a whole mouse brain can be imaged with x-ray microscopes without significant image degradation should appropriate image reconstruction methods be identified.« less

Electron Microscopy and Analytical X-ray Characterization of Compositional and Nanoscale Structural Changes in Fossil Bone

NASA Astrophysics Data System (ADS)

Boatman, Elizabeth Marie

The nanoscale structure of compact bone contains several features that are direct indicators of bulk tissue mechanical properties. Fossil bone tissues represent unique opportunities to understand the compact bone structure/property relationships from a deep time perspective, offering a possible array of new insights into bone diseases, biomimicry of composite materials, and basic knowledge of bioapatite composition and nanoscale bone structure. To date, most work with fossil bone has employed microscale techniques and has counter-indicated the survival of bioapatite and other nanoscale structural features. The obvious disconnect between the use of microscale techniques and the discernment of nanoscale structure has prompted this work. The goal of this study was to characterize the nanoscale constituents of fossil compact bone by applying a suite of diffraction, microscopy, and spectrometry techniques, representing the highest levels of spatial and energy resolution available today, and capable of complementary structural and compositional characterization from the micro- to the nanoscale. Fossil dinosaur and crocodile long bone specimens, as well as modern ratite and crocodile femurs, were acquired from the UC Museum of Paleontology. Preserved physiological features of significance were documented with scanning electron microscopy back-scattered imaging. Electron microprobe wavelength-dispersive X-ray spectroscopy (WDS) revealed fossil bone compositions enriched in fluorine with a complementary loss of oxygen. X-ray diffraction analyses demonstrated that all specimens were composed of apatite. Transmission electron microscopy (TEM) imaging revealed preserved nanocrystallinity in the fossil bones and electron diffraction studies further identified these nanocrystallites as apatite. Tomographic analyses of nanoscale elements imaged by TEM and small angle X-ray scattering were performed, with the results of each analysis further indicating that nanoscale structure is

High yield production of long branched Au nanoparticles characterized by atomic resolution transmission electron microscopy

PubMed Central

Mayoral, Alvaro; Magen, Cesar; Jose-Yacaman, Miguel

2011-01-01

Long multi-branched gold nanoparticles have been synthesized in a very high yield through a facile synthesis combining two different capping agents. The stability of these materials with the time has been tested and their characterization have been performed by diverse advanced electron microscopy techniques, paying special attention to aberration corrected transmission electron microscopy in order to unambiguously analyze the surface structure of the branches and provide insights for the formation of stellated gold nanoparticles. PMID:22125420

Electron probe X-ray microanalysis of cultured myogenic C2C12 cells with scanning and scanning transmission electron microscopy.

PubMed

Tylko, G; Karasiński, J; Wróblewski, R; Roomans, G M; Kilarski, W M

2000-01-01

Heterogeneity of the elemental content of myogenic C2C12 cultured cells was studied by electron probe X-ray microanalysis (EPXMA) with scanning (SEM EPXMA) and scanning transmission electron microscopy (STEM EPXMA). The best plastic substrate for growing cells was Thermanox. For STEM EPXMA, a Formvar film coated with carbon was found to be suitable substrate. The cells examined by scanning transmission electron microscopy showed great heterogeneity in their elemental content in comparison with the cells examined in the scanning electron microscope despite of an almost identical preparation procedure for EPXMA. Nevertheless the K/Na ratios obtained from both methods of EPXMA were very close (4.1 and 4.3). We conclude that the observed discrepancy in the elemental content obtained by the two methods may be due to differences in instrumentation and this must be taken into account when planning a comparative study.

Sea Spray Aerosol Structure and Composition Using Cryogenic Transmission Electron Microscopy

PubMed Central

2016-01-01

The composition and surface properties of atmospheric aerosol particles largely control their impact on climate by affecting their ability to uptake water, react heterogeneously, and nucleate ice in clouds. However, in the vacuum of a conventional electron microscope, the native surface and internal structure often undergo physicochemical rearrangement resulting in surfaces that are quite different from their atmospheric configurations. Herein, we report the development of cryogenic transmission electron microscopy where laboratory generated sea spray aerosol particles are flash frozen in their native state with iterative and controlled thermal and/or pressure exposures and then probed by electron microscopy. This unique approach allows for the detection of not only mixed salts, but also soft materials including whole hydrated bacteria, diatoms, virus particles, marine vesicles, as well as gel networks within hydrated salt droplets—all of which will have distinct biological, chemical, and physical processes. We anticipate this method will open up a new avenue of analysis for aerosol particles, not only for ocean-derived aerosols, but for those produced from other sources where there is interest in the transfer of organic or biological species from the biosphere to the atmosphere. PMID:26878061

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy.

PubMed

Höhn, K; Fuchs, J; Fröber, A; Kirmse, R; Glass, B; Anders-Össwein, M; Walther, P; Kräusslich, H-G; Dietrich, C

2015-08-01

In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV-pulsed mature human dendritic cells. © 2015 The Authors Journal of Microscopy © 2015 Royal Microscopical Society.

Click-electron microscopy for imaging metabolically tagged non-protein biomolecules

PubMed Central

Ngo, John T.; Adams, Stephen R.; Deerinck, Thomas J.; Boassa, Daniela; Rodriguez-Rivera, Frances; Palida, Sakina F.; Bertozzi, Carolyn R.; Ellisman, Mark H.; Tsien, Roger Y.

2016-01-01

Electron microscopy (EM) has long been the main technique to image cell structures with nanometer resolution, but has lagged behind light microscopy in the crucial ability to make specific molecules stand out. Here we introduce “Click-EM,” a labeling technique for correlative light microscopy and EM imaging of non-protein biomolecules. In this approach, metabolic labeling substrates containing bioorthogonal functional groups are provided to cells for incorporation into biopolymers by endogenous biosynthetic machinery. The unique chemical functionality of these analogs is exploited for selective attachment of singlet oxygen-generating fluorescent dyes via bioorthogonal “click chemistry” ligations. Illumination of dye-labeled structures generates singlet oxygen to locally catalyze the polymerization of diaminobenzidine into an osmiophilic reaction product that is readily imaged by EM. We describe the application of Click-EM in imaging metabolically tagged DNA, RNA, and lipids in cultured cells and neurons, and highlight its use in tracking peptidoglycan synthesis in the Gram-positive bacterium Listeria monocytogenes. PMID:27110681

Thickness determination of few-layer hexagonal boron nitride films by scanning electron microscopy and Auger electron spectroscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sutter, P., E-mail: psutter@bnl.gov; Sutter, E.

2014-09-01

We assess scanning electron microscopy (SEM) and Auger electron spectroscopy (AES) for thickness measurements on few-layer hexagonal boron nitride (h-BN), the layered dielectric of choice for integration with graphene and other two-dimensional materials. Observations on h-BN islands with large, atomically flat terraces show that the secondary electron intensity in SEM reflects monolayer height changes in films up to least 10 atomic layers thickness. From a quantitative analysis of AES data, the energy-dependent electron escape depth in h-BN films is deduced. The results show that AES is suitable for absolute thickness measurements of few-layer h-BN of 1 to 6 layers.

Robust image alignment for cryogenic transmission electron microscopy.

PubMed

McLeod, Robert A; Kowal, Julia; Ringler, Philippe; Stahlberg, Henning

2017-03-01

Cryo-electron microscopy recently experienced great improvements in structure resolution due to direct electron detectors with improved contrast and fast read-out leading to single electron counting. High frames rates enabled dose fractionation, where a long exposure is broken into a movie, permitting specimen drift to be registered and corrected. The typical approach for image registration, with high shot noise and low contrast, is multi-reference (MR) cross-correlation. Here we present the software package Zorro, which provides robust drift correction for dose fractionation by use of an intensity-normalized cross-correlation and logistic noise model to weight each cross-correlation in the MR model and filter each cross-correlation optimally. Frames are reliably registered by Zorro with low dose and defocus. Methods to evaluate performance are presented, by use of independently-evaluated even- and odd-frame stacks by trajectory comparison and Fourier ring correlation. Alignment of tiled sub-frames is also introduced, and demonstrated on an example dataset. Zorro source code is available at github.com/CINA/zorro. Copyright © 2016 Elsevier Inc. All rights reserved.

Advanced electron microscopy characterization of nanomaterials for catalysis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Su, Dong

Transmission electron microscopy (TEM) has become one of the most powerful techniques in the fields of material science, inorganic chemistry and nanotechnology. In terms of resolutions, advanced TEM may reach a high spatial resolution of 0.05 nm, a high energy-resolution of 7 meV. In addition, in situ TEM can help researcher to image the process happened within 1 ms. This paper reviews the recent technical approaches of applying advanced TEM characterization on nanomaterials for catalysis. The text is organized according to the demanded information of nanocrystals from the perspective of application: for example, size, composition, phase, strain, and morphology. Themore » electron beam induced effect and in situ TEM are also introduced. As a result, I hope this review can help the scientists in related fields to take advantage of advanced TEM to their own researches.« less

Advanced electron microscopy characterization of nanomaterials for catalysis

DOE PAGES

Su, Dong

2017-04-01

Transmission electron microscopy (TEM) has become one of the most powerful techniques in the fields of material science, inorganic chemistry and nanotechnology. In terms of resolutions, advanced TEM may reach a high spatial resolution of 0.05 nm, a high energy-resolution of 7 meV. In addition, in situ TEM can help researcher to image the process happened within 1 ms. This paper reviews the recent technical approaches of applying advanced TEM characterization on nanomaterials for catalysis. The text is organized according to the demanded information of nanocrystals from the perspective of application: for example, size, composition, phase, strain, and morphology. Themore » electron beam induced effect and in situ TEM are also introduced. As a result, I hope this review can help the scientists in related fields to take advantage of advanced TEM to their own researches.« less

The role of electron irradiation history in liquid cell transmission electron microscopy.

PubMed

Moser, Trevor H; Mehta, Hardeep; Park, Chiwoo; Kelly, Ryan T; Shokuhfar, Tolou; Evans, James E

2018-04-01

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.

The role of electron irradiation history in liquid cell transmission electron microscopy

PubMed Central

Mehta, Hardeep

2018-01-01

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides. PMID:29725619

The role of electron irradiation history in liquid cell transmission electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC- TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the rolemore » of cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. These results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

Electron Microscopy Localization and Characterization of Functionalized Composite Organic-Inorganic SERS Nanoparticles on Leukemia Cells

PubMed Central

Koh, Ai Leen; Shachaf, Catherine M.; Elchuri, Sailaja; Nolan, Garry P.; Sinclair, Robert

2008-01-01

We demonstrate the use of electron microscopy as a powerful characterization tool to identify and locate antibody-conjugated composite organic-inorganic (COINs) surface enhanced Raman scattering (SERS) nanoparticles on cells. U937 leukemia cells labeled with antibody CD54-conjugated COINs were characterized in their native, hydrated state using wet Scanning Electron Microscopy (SEM) and in their dehydrated state using high-resolution SEM. In both cases, the backscattered electron detector (BSE) was used to detect and identify the silver constituents in COINs due to its high sensitivity to atomic number variations within a specimen. The imaging and analytical capabilities in the SEM were further complemented by higher resolution Transmission Electron Microscope (TEM) images and Scanning Auger Electron Spectroscopy (AES) data to give reliable and high-resolution information about nanoparticles and their binding to cell surface antigens. PMID:18995965

Investigation of nanoparticulate silicon as printed layers using scanning electron microscopy, transmission electron microscopy, X-ray absorption spectroscopy and X-ray photoelectron spectroscopy

DOE PAGES

Unuigbe, David M.; Harting, Margit; Jonah, Emmanuel O.; ...

2017-08-21

The presence of native oxide on the surface of silicon nanoparticles is known to inhibit charge transport on the surfaces. Scanning electron microscopy (SEM) studies reveal that the particles in the printed silicon network have a wide range of sizes and shapes. High-resolution transmission electron microscopy reveals that the particle surfaces have mainly the (111)- and (100)-oriented planes which stabilizes against further oxidation of the particles. X-ray absorption spectroscopy (XANES) and X-ray photoelectron spectroscopy (XPS) measurements at the O 1s-edge have been utilized to study the oxidation and local atomic structure of printed layers of silicon nanoparticles which were milledmore » for different times. XANES results reveal the presence of the +4 (SiO 2) oxidation state which tends towards the +2 (SiO) state for higher milling times. Si 2pXPS results indicate that the surfaces of the silicon nanoparticles in the printed layers are only partially oxidized and that all three sub-oxide, +1 (Si 2O), +2 (SiO) and +3 (Si 2O 3), states are present. The analysis of the change in the sub-oxide peaks of the silicon nanoparticles shows the dominance of the +4 state only for lower milling times.« less

Fixation methods for electron microscopy of human and other liver

PubMed Central

Wisse, Eddie; Braet, Filip; Duimel, Hans; Vreuls, Celien; Koek, Ger; Olde Damink, Steven WM; van den Broek, Maartje AJ; De Geest, Bart; Dejong, Cees HC; Tateno, Chise; Frederik, Peter

2010-01-01

For an electron microscopic study of the liver, expertise and complicated, time-consuming processing of hepatic tissues and cells is needed. The interpretation of electron microscopy (EM) images requires knowledge of the liver fine structure and experience with the numerous artifacts in fixation, embedding, sectioning, contrast staining and microscopic imaging. Hence, the aim of this paper is to present a detailed summary of different methods for the preparation of hepatic cells and tissue, for the purpose of preserving long-standing expertise and to encourage new investigators and clinicians to include EM studies of liver cells and tissue in their projects. PMID:20556830

Preservation of protein fluorescence in embedded human dendritic cells for targeted 3D light and electron microscopy

PubMed Central

HÖHN, K.; FUCHS, J.; FRÖBER, A.; KIRMSE, R.; GLASS, B.; ANDERS‐ÖSSWEIN, M.; WALTHER, P.; KRÄUSSLICH, H.‐G.

2015-01-01

Summary In this study, we present a correlative microscopy workflow to combine detailed 3D fluorescence light microscopy data with ultrastructural information gained by 3D focused ion beam assisted scanning electron microscopy. The workflow is based on an optimized high pressure freezing/freeze substitution protocol that preserves good ultrastructural detail along with retaining the fluorescence signal in the resin embedded specimens. Consequently, cellular structures of interest can readily be identified and imaged by state of the art 3D confocal fluorescence microscopy and are precisely referenced with respect to an imprinted coordinate system on the surface of the resin block. This allows precise guidance of the focused ion beam assisted scanning electron microscopy and limits the volume to be imaged to the structure of interest. This, in turn, minimizes the total acquisition time necessary to conduct the time consuming ultrastructural scanning electron microscope imaging while eliminating the risk to miss parts of the target structure. We illustrate the value of this workflow for targeting virus compartments, which are formed in HIV‐pulsed mature human dendritic cells. PMID:25786567

Rumen Bacterial Degradation of Forage Cell Walls Investigated by Electron Microscopy

PubMed Central

Akin, Danny E.; Amos, Henry E.

1975-01-01

The association of rumen bacteria with specific leaf tissues of the forage grass Kentucky-31 tall fescue (Festuca arundinacea Schreb.) during in vitro degradation was investigated by transmission and scanning electron microscopy. Examination of degraded leaf cross-sections revealed differential rates of tissue degradation in that the cell walls of the mesophyll and pholem were degraded prior to those of the outer bundle sheath and epidermis. Rumen bacteria appeared to degrade the mesophyll, in some cases, and phloem without prior attachment to the plant cell walls. The degradation of bundle sheath and epidermal cell walls appeared to be preceded by attachment of bacteria to the plant cell wall. Ultrastructural features apparently involved in the adhesion of large cocci to plant cells were observed by transmission and scanning electron microscopy. The physical association between plant and rumen bacterial cells during degradation apparently varies with tissue types. Bacterial attachment, by extracellular features in some microorganisms, is required prior to degradation of the more resistant tissues. Images PMID:16350017

Reflections on the value of electron microscopy in the study of heterogeneous catalysts

PubMed Central

2017-01-01

Electron microscopy (EM) is arguably the single most powerful method of characterizing heterogeneous catalysts. Irrespective of whether they are bulk and multiphasic, or monophasic and monocrystalline, or nanocluster and even single-atom and on a support, their structures in atomic detail can be visualized in two or three dimensions, thanks to high-resolution instruments, with sub-Ångstrom spatial resolutions. Their topography, tomography, phase-purity, composition, as well as the bonding, and valence-states of their constituent atoms and ions and, in favourable circumstances, the short-range and long-range atomic order and dynamics of the catalytically active sites, can all be retrieved by the panoply of variants of modern EM. The latter embrace electron crystallography, rotation and precession electron diffraction, X-ray emission and high-resolution electron energy-loss spectra (EELS). Aberration-corrected (AC) transmission (TEM) and scanning transmission electron microscopy (STEM) have led to a revolution in structure determination. Environmental EM is already playing an increasing role in catalyst characterization, and new advances, involving special cells for the study of solid catalysts in contact with liquid reactants, have recently been deployed. PMID:28265196

High-quality ultrastructural preservation using cryofixation for 3D electron microscopy of genetically labeled tissues

PubMed Central

Boassa, Daniela; Hu, Junru; Romoli, Benedetto; Phan, Sebastien; Dulcis, Davide

2018-01-01

Electron microscopy (EM) offers unparalleled power to study cell substructures at the nanoscale. Cryofixation by high-pressure freezing offers optimal morphological preservation, as it captures cellular structures instantaneously in their near-native state. However, the applicability of cryofixation is limited by its incompatibility with diaminobenzidine labeling using genetic EM tags and the high-contrast en bloc staining required for serial block-face scanning electron microscopy (SBEM). In addition, it is challenging to perform correlated light and electron microscopy (CLEM) with cryofixed samples. Consequently, these powerful methods cannot be applied to address questions requiring optimal morphological preservation. Here, we developed an approach that overcomes these limitations; it enables genetically labeled, cryofixed samples to be characterized with SBEM and 3D CLEM. Our approach is broadly applicable, as demonstrated in cultured cells, Drosophila olfactory organ and mouse brain. This optimization exploits the potential of cryofixation, allowing for quality ultrastructural preservation for diverse EM applications. PMID:29749931

Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy.

PubMed

Nannenga, Brent L; Iadanza, Matthew G; Vollmar, Breanna S; Gonen, Tamir

2013-01-01

Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.

STEM VQ Method, Using Scanning Transmission Electron Microscopy (STEM) for Accurate Virus Quantification

DTIC Science & Technology

2017-02-02

Corresponding Author Abstract Accurate virus quantification is sought, but a perfect method still eludes the scientific community. Electron...unlimited. UNCLASSIFIED 2 provides morphology data and counts all viral particles, including partial or noninfectious particles; however, EM methods ...consistent, reproducible virus quantification method called Scanning Transmission Electron Microscopy – Virus Quantification (STEM-VQ) which simplifies

Electronic cameras for low-light microscopy.

PubMed

Rasnik, Ivan; French, Todd; Jacobson, Ken; Berland, Keith

2013-01-01

This chapter introduces to electronic cameras, discusses the various parameters considered for evaluating their performance, and describes some of the key features of different camera formats. The chapter also presents the basic understanding of functioning of the electronic cameras and how these properties can be exploited to optimize image quality under low-light conditions. Although there are many types of cameras available for microscopy, the most reliable type is the charge-coupled device (CCD) camera, which remains preferred for high-performance systems. If time resolution and frame rate are of no concern, slow-scan CCDs certainly offer the best available performance, both in terms of the signal-to-noise ratio and their spatial resolution. Slow-scan cameras are thus the first choice for experiments using fixed specimens such as measurements using immune fluorescence and fluorescence in situ hybridization. However, if video rate imaging is required, one need not evaluate slow-scan CCD cameras. A very basic video CCD may suffice if samples are heavily labeled or are not perturbed by high intensity illumination. When video rate imaging is required for very dim specimens, the electron multiplying CCD camera is probably the most appropriate at this technological stage. Intensified CCDs provide a unique tool for applications in which high-speed gating is required. The variable integration time video cameras are very attractive options if one needs to acquire images at video rate acquisition, as well as with longer integration times for less bright samples. This flexibility can facilitate many diverse applications with highly varied light levels. Copyright © 2007 Elsevier Inc. All rights reserved.

Use of fluorescence and scanning electron microscopy as tools in teaching biology

NASA Astrophysics Data System (ADS)

Ghosh, Nabarun; Silva, Jessica; Vazquez, Aracely; Das, A. B.; Smith, Don W.

2011-06-01

Recent nationwide surveys reveal significant decline in students' interest in Math and Sciences. The objective of this project was to inspire young minds in using various techniques involved in Sciences including Scanning Electron Microscopy. We used Scanning Electron Microscope in demonstrating various types of Biological samples. An SEM Tabletop model in the past decade has revolutionized the use of Scanning Electron Microscopes. Using SEM Tabletop model TM 1000 we studied biological specimens of fungal spores, pollen grains, diatoms, plant fibers, dust mites, insect parts and leaf surfaces. We also used fluorescence microscopy to view, to record and analyze various specimens with an Olympus BX40 microscope equipped with FITC and TRITC fluorescent filters, a mercury lamp source, DP-70 digital camera with Image Pro 6.0 software. Micrographs were captured using bright field microscopy, the fluoresceinisothiocyanate (FITC) filter, and the tetramethylrhodamine (TRITC) filter settings at 40X. A high pressure mercury lamp or UV source was used to excite the storage molecules or proteins which exhibited autofluorescence. We used fluorescent microscopy to confirm the localization of sugar beet viruses in plant organs by viewing the vascular bundles in the thin sections of the leaves and other tissues. We worked with the REU summer students on sample preparation and observation on various samples utilizing the SEM. Critical Point Drying (CPD) and metal coating with the sputter coater was followed before observing some cultured specimen and the samples that were soft in textures with high water content. SEM Top allowed investigating the detailed morphological features that can be used for classroom teaching. Undergraduate and graduate researchers studied biological samples of Arthropods, pollen grains and teeth collected from four species of snakes using SEM. This project inspired the research students to pursue their career in higher studies in science and 45% of the

Contamination mitigation strategies for scanning transmission electron microscopy.

PubMed

Mitchell, D R G

2015-06-01

Modern scanning transmission electron microscopy (STEM) enables imaging and microanalysis at very high magnification. In the case of aberration-corrected STEM, atomic resolution is readily achieved. However, the electron fluxes used may be up to three orders of magnitude greater than those typically employed in conventional STEM. Since specimen contamination often increases with electron flux, specimen cleanliness is a critical factor in obtaining meaningful data when carrying out high magnification STEM. A range of different specimen cleaning methods have been applied to a variety of specimen types. The contamination rate has been measured quantitatively to assess the effectiveness of cleaning. The methods studied include: baking, cooling, plasma cleaning, beam showering and UV/ozone exposure. Of the methods tested, beam showering is rapid, experimentally convenient and very effective on a wide range of specimens. Oxidative plasma cleaning is also very effective and can be applied to specimens on carbon support films, albeit with some care. For electron beam-sensitive materials, cooling may be the method of choice. In most cases, preliminary removal of the bulk of the contamination by methods such as baking or plasma cleaning, followed by beam showering, where necessary, can result in a contamination-free specimen suitable for extended atomic scale imaging and analysis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Transmission Electron Microscopy Analysis of Skin Lesions from Sporotrichosis Epidemic in Rio de Janeiro, Brazil

PubMed Central

Porto Ferreira, Cassio; Oliveira de Almeida, Ana Cristina; Corte-Real, Suzana

2015-01-01

Transmission electron microscopy can yield useful information in a range of scientific fields; it is capable of imaging at a significantly higher resolution than light microscopes and has been a very useful tool in the identification of morphological changes of the dermis as well as assessment of changes in the extracellular matrix. Our aim is to characterize by electron microscopy the cellular profile of lesions caused by Sporothrix schenckii from the sporotrichosis epidemic in its zoonotic form that occurs in Rio de Janeiro, Brazil. PMID:25653392

Tissue and cellular localization of tannins in Tunisian dates (Phoenix dactylifera L.) by light and transmission electron microscopy.

PubMed

Hammouda, Hédi; Alvarado, Camille; Bouchet, Brigitte; Kalthoum-Chérif, Jamila; Trabelsi-Ayadi, Malika; Guyot, Sylvain

2014-07-16

A histological approach including light microscopy and transmission electron microscopy (TEM) was used to provide accurate information on the localization of condensed tannins in the edible tissues and in the stone of date fruits (Phoenix dactylifera L.). Light microscopy was carried out on fresh tissues after staining by 4-dimethylaminocinnamaldehyde (DMACA) for a specific detection of condensed tannins. Thus, whether under light microscopy or transmission electron microscopy (TEM), results showed that tannins are not located in the epidermis but more deeply in the mesocarp in the vacuole of very large cells. Regarding the stones, tannins are found in a specific cell layer located at 50 μm from the sclereid cells of the testa.

Fundamental Technical Elements of Freeze-fracture/Freeze-etch in Biological Electron Microscopy

EPA Science Inventory

Freeze-fracture/freeze-etch describes a process whereby specimens, typically biological or nanomaterial in nature, are frozen, fractured, and replicated to generate a carbon/platinum "cast" intended for examination by transmission electron microscopy. Specimens are subjected to u...

U-10Mo Sample Preparation and Examination using Optical and Scanning Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prabhakaran, Ramprashad; Joshi, Vineet V.; Rhodes, Mark A.

2016-10-01

The purpose of this document is to provide guidelines to prepare specimens of uranium alloyed with 10 weight percent molybdenum (U-10Mo) for optical metallography and scanning electron microscopy. This document also provides instructions to set up an optical microscope and a scanning electron microscope to analyze U-10Mo specimens and to obtain the required information.

U-10Mo Sample Preparation and Examination using Optical and Scanning Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prabhakaran, Ramprashad; Joshi, Vineet V.; Rhodes, Mark A.

2016-03-30

The purpose of this document is to provide guidelines to prepare specimens of uranium alloyed with 10 weight percent molybdenum (U-10Mo) for optical metallography and scanning electron microscopy. This document also provides instructions to set up an optical microscope and a scanning electron microscope to analyze U-10Mo specimens and to obtain the required information.

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

PubMed Central

Moriya, Toshio; Saur, Michael; Stabrin, Markus; Merino, Felipe; Voicu, Horatiu; Huang, Zhong; Penczek, Pawel A.; Raunser, Stefan; Gatsogiannis, Christos

2017-01-01

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 Å structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state. PMID:28570515

Atomic force microscopy and transmission electron microscopy analyses of low-temperature laser welding of the cornea.

PubMed

Matteini, Paolo; Sbrana, Francesca; Tiribilli, Bruno; Pini, Roberto

2009-07-01

Low-temperature laser welding of the cornea is a technique used to facilitate the closure of corneal cuts. The procedure consists of staining the wound with a chromophore (indocyanine green), followed by continuous wave irradiation with an 810 nm diode laser operated at low power densities (12-16 W/cm(2)), which induces local heating in the 55-65 degrees C range. In this study, we aimed to investigate the ultrastructural modifications in the extracellular matrix following laser welding of corneal wounds by means of atomic force microscopy and transmission electron microscopy. The results evidenced marked disorganization of the normal fibrillar assembly, although collagen appeared not to be denatured under the operating conditions we employed. The mechanism of low-temperature laser welding may be related to some structural modifications of the nonfibrillar extracellular components of the corneal stroma.

Magnetism of epitaxial Tb films on W(110) studied by spin-polarized low-energy electron microscopy

NASA Astrophysics Data System (ADS)

Prieto, J. E.; Chen, Gong; Schmid, A. K.; de la Figuera, J.

2016-11-01

Thin epitaxial films of Tb metal were grown on a clean W(110) substrate in ultrahigh vacuum and studied in situ by low-energy electron microscopy. Annealed films present magnetic contrast in spin-polarized low-energy electron microscopy. The energy dependence of the electron reflectivity was determined and a maximum value of its spin asymmetry of about 1% was measured. The magnetization direction of the Tb films is in-plane. Upon raising the temperature, no change in the domain distribution is observed, while the asymmetry in the electron reflectivity decreases when approaching the critical temperature, following a power law ˜(1-T /TC) β with a critical exponent β of 0.39.

Amyloid Structure and Assembly: Insights from Scanning Transmission Electron Microscopy

PubMed Central

Goldsbury, Claire; Baxa, Ulrich; Simon, Martha N.; Steven, Alasdair C.; Engel, Andreas; Wall, Joseph S.; Aebi, Ueli; Müller, Shirley A.

2010-01-01

Amyloid fibrils are filamentous protein aggregates implicated in several common diseases like Alzheimer’s disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies like Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR). PMID:20868754

Investigation of porous asphalt microstructure using optical and electron microscopy.

PubMed

Poulikakos, L D; Partl, M N

2010-11-01

Direct observations of porous asphalt concrete samples in their natural state using optical and electron microscopy techniques led to useful information regarding the microstructure of two mixes and indicated a relationship between microstructure and in situ performance. This paper presents evidence that suboptimal microstructure can lead to premature failure thus making a first step in defining well or suboptimal performing pavements with a bottom-up approach (microstructure). Laboratory and field compaction produce different samples in terms of the microstructure. Laboratory compaction using the gyratory method has produced more microcracks in mineral aggregates after the binder had cooled. Well-performing mixes used polymer-modified binders, had a more homogeneous void structure with fewer elongated voids and better interlocking of the aggregates. Furthermore, well-performing mixes showed better distribution of the mastic and better coverage of the aggregates with bitumen. Low vacuum scanning electron microscopy showed that styrene butadiene styrene polymer modification in binder exists in the form of discontinuous globules and not continuous networks. A reduction in the polymer phase was observed as a result of aging and in-service use. © 2010 The Authors Journal compilation © 2010 The Royal Microscopical Society.

Solution and electron microscopy characterization of lactococcal phage baseplates expressed in Escherichia coli.

PubMed

Campanacci, Valérie; Veesler, David; Lichière, Julie; Blangy, Stéphanie; Sciara, Giuliano; Moineau, Sylvain; van Sinderen, Douwe; Bron, Patrick; Cambillau, Christian

2010-10-01

We report here the characterization of several large structural protein complexes forming the baseplates (or part of them) of Siphoviridae phages infecting Lactococcus lactis: TP901-1, Tuc2009 and p2. We revisited a "block cloning" expression strategy and extended this approach to genomic fragments encoding proteins whose interacting partners have not yet been clearly identified. Biophysical characterization of some of these complexes using circular dichroism and size exclusion chromatography, coupled with on-line light scattering and refractometry, demonstrated that the over-produced recombinant proteins interact with each other to form large (up to 1.9MDa) and stable baseplate assemblies. Some of these complexes were characterized by electron microscopy confirming their structural homogeneity as well as providing a picture of their overall molecular shapes and symmetry. Finally, using these results, we were able to highlight similarities and differences with the well characterized much larger baseplate of the myophage T4.

A literature review of in situ transmission electron microscopy technique in corrosion studies.

PubMed

Song, Zhengwei; Xie, Zhi-Hui

2018-06-18

One of the biggest challenges in corrosion investigation is foreseeing precisely how and where materials will degenerate in a designated condition owing to scarceness of accurate corrosion mechanisms. Recent fast development of in situ transmission electron microscopy (TEM) technique makes it achievable to better understand the corrosion mechanism and physicochemical processes at the interfaces between samples and gases or electrolytes by dynamical capture the microstructural and chemical changes with high resolution within a realistic or near-realistic environment. However, a detailed and in-depth account summing up the development and latest achievements of in situ TEM techniques, especially the application of emerging liquid and electrochemical cells in the community of corrosion study in the last several years is lacking and is urgently needed for its heathy development. To fill this gap, this critical review summarizes firstly the key scientific issues in corrosion research, followed by introducing the configurations of several typical closed-type cells. Then, the achievements of in situ TEM using open-type or closed-type cells in corrosion study are presented in detail. The study directions in the future are commented finally in terms of spatial and temporal resolution, electron radiation, and linkage between microstructure and electrochemical performance in corrosion community. Copyright © 2018 Elsevier Ltd. All rights reserved.

Synthesis and Cs-Corrected Scanning Transmission Electron Microscopy Characterization of Multimetallic Nanoparticles

NASA Astrophysics Data System (ADS)

Khanal, Subarna; Bhattarai, Nabraj; Velázquez-Salazar, Jesus; Jose-Yacaman, Miguel; Subarna Khanal Team

2014-03-01

Multimetallic nanoparticles have been attracted greater attention both in materials science and nanotechnology due to its unique electronic, optical, biological, and catalytic properties lead by physiochemical interactions among different atoms and phases. The distinct features of multimetallic nanoparticles enhanced synergetic properties, large surface to volume ratio and quantum size effects ultimately lead to novel and wide range of possibilities for different applications than monometallic counterparts. For instance, PtPd, Pt/Cu, Au-Au3Cu, AgPd/Pt, AuCu/Pt and many other multimetallic nanoparticles have raised interest for their various applications in fuel cells, ethanol and methanol oxidation reactions, hydrogen storage, and so on. The nanostructures were analyzed by transmission electron microscopy (TEM) and by aberration-corrected scanning transmission electron microscopy (Cs-corrected STEM), in combination with high angle annular dark field (HAADF), bright field (BF), energy dispersive X-ray spectroscopy (EDS), and electron energy loss spectroscopy (EELS) detectors. These techniques allowed us to probe the structure at the atomic level of the nanoparticles revealing new structural information and elemental composition of the nanoparticles. The authors would like to acknowledge NSF grants DMR-1103730, ``Alloys at the Nanoscale: The Case of Nanoparticles Second Phase'' and NSF PREM Grant # DMR 0934218.

Transmission electron microscopy analysis of skin lesions from sporotrichosis epidemic in Rio de Janeiro, Brazil.

PubMed

Ferreira, Cassio Porto; Oliveira de Almeida, Ana Cristina; Corte-Real, Suzana

2015-02-01

Transmission electron microscopy can yield useful information in a range of scientific fields; it is capable of imaging at a significantly higher resolution than light microscopes and has been a very useful tool in the identification of morphological changes of the dermis as well as assessment of changes in the extracellular matrix. Our aim is to characterize by electron microscopy the cellular profile of lesions caused by Sporothrix schenckii from the sporotrichosis epidemic in its zoonotic form that occurs in Rio de Janeiro, Brazil. © The American Society of Tropical Medicine and Hygiene.

Automated in-chamber specimen coating for serial block-face electron microscopy.

PubMed

Titze, B; Denk, W

2013-05-01

When imaging insulating specimens in a scanning electron microscope, negative charge accumulates locally ('sample charging'). The resulting electric fields distort signal amplitude, focus and image geometry, which can be avoided by coating the specimen with a conductive film prior to introducing it into the microscope chamber. This, however, is incompatible with serial block-face electron microscopy (SBEM), where imaging and surface removal cycles (by diamond knife or focused ion beam) alternate, with the sample remaining in place. Here we show that coating the sample after each cutting cycle with a 1-2 nm metallic film, using an electron beam evaporator that is integrated into the microscope chamber, eliminates charging effects for both backscattered (BSE) and secondary electron (SE) imaging. The reduction in signal-to-noise ratio (SNR) caused by the film is smaller than that caused by the widely used low-vacuum method. Sample surfaces as large as 12 mm across were coated and imaged without charging effects at beam currents as high as 25 nA. The coatings also enabled the use of beam deceleration for non-conducting samples, leading to substantial SNR gains for BSE contrast. We modified and automated the evaporator to enable the acquisition of SBEM stacks, and demonstrated the acquisition of stacks of over 1000 successive cut/coat/image cycles and of stacks using beam deceleration or SE contrast. © 2013 The Authors Journal of Microscopy © 2013 Royal Microscopical Society.

Droplet Epitaxy Image Contrast in Mirror Electron Microscopy

NASA Astrophysics Data System (ADS)

Kennedy, S. M.; Zheng, C. X.; Jesson, D. E.

2017-01-01

Image simulation methods are applied to interpret mirror electron microscopy (MEM) images obtained from a movie of GaAs droplet epitaxy. Cylindrical symmetry of structures grown by droplet epitaxy is assumed in the simulations which reproduce the main features of the experimental MEM image contrast, demonstrating that droplet epitaxy can be studied in real-time. It is therefore confirmed that an inner ring forms at the droplet contact line and an outer ring (or skirt) occurs outside the droplet periphery. We believe that MEM combined with image simulations will be increasingly used to study the formation and growth of quantum structures.

Development of a fountain detector for spectroscopy of secondary electrons in scanning electron microscopy

NASA Astrophysics Data System (ADS)

Agemura, Toshihide; Kimura, Takashi; Sekiguchi, Takashi

2018-04-01

The low-pass secondary electron (SE) detector, the so-called “fountain detector (FD)”, for scanning electron microscopy has high potential for application to the imaging of low-energy SEs. Low-energy SE imaging may be used for detecting the surface potential variations of a specimen. However, the detected SEs include a certain fraction of tertiary electrons (SE3s) because some of the high-energy backscattered electrons hit the grid to yield SE3s. We have overcome this difficulty by increasing the aperture ratio of the bias and ground grids and using the lock-in technique, in which the AC field with the DC offset was applied on the bias grid. The energy-filtered SE images of a 4H-SiC p-n junction show complex behavior according to the grid bias. These observations are clearly explained by the variations of Auger spectra across the p-n junction. The filtered SE images taken with the FD can be applied to observing the surface potential variation of specimens.

Diffusive and inelastic scattering in ballistic-electron-emission spectroscopy and ballistic-electron-emission microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, E.Y.; Turner, B.R.; Schowalter, L.J.

1993-07-01

Ballistic-electron-emission microscopy (BEEM) of Au/Si(001) n type was done to study whether elastic scattering in the Au overlayer is dominant. It was found that there is no dependence of the BEEM current on the relative gradient of the Au surface with respect to the Si interface, and this demonstrates that significant elastic scattering must occur in the Au overlayer. Ballistic-electron-emission spectroscopy (BEES) was also done, and, rather than using the conventional direct-current BEES, alternating-current (ac) BEES was done on Au/Si and also on Au/PtSi/Si(001) n type. The technique of ac BEES was found to give linear threshold for the Schottkymore » barrier, and it also clearly showed the onset of electron-hole pair creation and other inelastic scattering events. The study of device quality PtSi in Au/PtSi/Si(001) yielded an attenuation length of 4 nm for electrons of energy 1 eV above the PtSi Fermi energy. 20 refs., 5 figs.« less

The role of electron irradiation history in liquid cell transmission electron microscopy

DOE PAGES

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo; ...

2018-04-20

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role ofmore » cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. Lastly, these results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

The role of electron irradiation history in liquid cell transmission electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moser, Trevor H.; Mehta, Hardeep; Park, Chiwoo

In situ liquid cell transmission electron microscopy (LC-TEM) allows dynamic nanoscale characterization of systems in a hydrated state. Although powerful, this technique remains impaired by issues of repeatability that limit experimental fidelity and hinder the identification and control of some variables underlying observed dynamics. We detail new LC-TEM devices that improve experimental reproducibility by expanding available imaging area and providing a platform for investigating electron flux history on the sample. Irradiation history is an important factor influencing LC-TEM results that has, to this point, been largely qualitatively and not quantitatively described. We use these devices to highlight the role ofmore » cumulative electron flux history on samples from both nanoparticle growth and biological imaging experiments and demonstrate capture of time zero, low-dose images on beam-sensitive samples. In particular, the ability to capture pristine images of biological samples, where the acquired image is the first time that the cell experiences significant electron flux, allowed us to determine that nanoparticle movement compared to the cell membrane was a function of cell damage and therefore an artifact rather than visualizing cell dynamics in action. Lastly, these results highlight just a subset of the new science that is accessible with LC-TEM through the new multiwindow devices with patterned focusing aides.« less

Specimen preparation by ion beam slope cutting for characterization of ductile damage by scanning electron microscopy.

PubMed

Besserer, Hans-Bernward; Gerstein, Gregory; Maier, Hans Jürgen; Nürnberger, Florian

2016-04-01

To investigate ductile damage in parts made by cold sheet-bulk metal forming a suited specimen preparation is required to observe the microstructure and defects such as voids by electron microscopy. By means of ion beam slope cutting both a targeted material removal can be applied and mechanical or thermal influences during preparation avoided. In combination with scanning electron microscopy this method allows to examine voids in the submicron range and thus to analyze early stages of ductile damage. In addition, a relief structure is formed by the selectivity of the ion bombardment, which depends on grain orientation and microstructural defects. The formation of these relief structures is studied using scanning electron microscopy and electron backscatter diffraction and the use of this side effect to interpret the microstructural mechanisms of voids formation by plastic deformation is discussed. A comprehensive investigation of the suitability of ion beam milling to analyze ductile damage is given at the examples of a ferritic deep drawing steel and a dual phase steel. © 2016 Wiley Periodicals, Inc.

Cryo-electron microscopy and cryo-electron tomography of nanoparticles.

PubMed

Stewart, Phoebe L

2017-03-01

Cryo-transmission electron microscopy (cryo-TEM or cryo-EM) and cryo-electron tomography (cryo-ET) offer robust and powerful ways to visualize nanoparticles. These techniques involve imaging of the sample in a frozen-hydrated state, allowing visualization of nanoparticles essentially as they exist in solution. Cryo-TEM grid preparation can be performed with the sample in aqueous solvents or in various organic and ionic solvents. Two-dimensional (2D) cryo-TEM provides a direct way to visualize the polydispersity within a nanoparticle preparation. Fourier transforms of cryo-TEM images can confirm the structural periodicity within a sample. While measurement of specimen parameters can be performed with 2D TEM images, determination of a three-dimensional (3D) structure often facilitates more spatially accurate quantization. 3D structures can be determined in one of two ways. If the nanoparticle has a homogeneous structure, then 2D projection images of different particles can be averaged using a computational process referred to as single particle reconstruction. Alternatively, if the nanoparticle has a heterogeneous structure, then a structure can be generated by cryo-ET. This involves collecting a tilt-series of 2D projection images for a defined region of the grid, which can be used to generate a 3D tomogram. Occasionally it is advantageous to calculate both a single particle reconstruction, to reveal the regular portions of a nanoparticle structure, and a cryo-electron tomogram, to reveal the irregular features. A sampling of 2D cryo-TEM images and 3D structures are presented for protein based, DNA based, lipid based, and polymer based nanoparticles. WIREs Nanomed Nanobiotechnol 2017, 9:e1417. doi: 10.1002/wnan.1417 For further resources related to this article, please visit the WIREs website. © 2016 Wiley Periodicals, Inc.

Reciprocity relations in transmission electron microscopy: A rigorous derivation.

PubMed

Krause, Florian F; Rosenauer, Andreas

2017-01-01

A concise derivation of the principle of reciprocity applied to realistic transmission electron microscopy setups is presented making use of the multislice formalism. The equivalence of images acquired in conventional and scanning mode is thereby rigorously shown. The conditions for the applicability of the found reciprocity relations is discussed. Furthermore the positions of apertures in relation to the corresponding lenses are considered, a subject which scarcely has been addressed in previous publications. Copyright © 2016 Elsevier Ltd. All rights reserved.

Practical aspects of monochromators developed for transmission electron microscopy

PubMed Central

Kimoto, Koji

2014-01-01

A few practical aspects of monochromators recently developed for transmission electron microscopy are briefly reviewed. The basic structures and properties of four monochromators, a single Wien filter monochromator, a double Wien filter monochromator, an omega-shaped electrostatic monochromator and an alpha-shaped magnetic monochromator, are outlined. The advantages and side effects of these monochromators in spectroscopy and imaging are pointed out. A few properties of the monochromators in imaging, such as spatial or angular chromaticity, are also discussed. PMID:25125333

Assessment of occupational exposure to asbestos fibers: Contribution of analytical transmission electron microscopy analysis and comparison with phase-contrast microscopy.

PubMed

Eypert-Blaison, Céline; Romero-Hariot, Anita; Clerc, Frédéric; Vincent, Raymond

2018-03-01

From November 2009 to October 2010, the French general directorate for labor organized a large field-study using analytical transmission electron microscopy (ATEM) to characterize occupational exposure to asbestos fibers during work on asbestos containing materials (ACM). The primary objective of this study was to establish a method and to validate the feasibility of using ATEM for the analysis of airborne asbestos of individual filters sampled in various occupational environments. For each sampling event, ATEM data were compared to those obtained by phase-contrast optical microscopy (PCOM), the WHO-recommended reference technique. A total of 265 results were obtained from 29 construction sites where workers were in contact with ACM. Data were sorted depending on the combination of the ACM type and the removal technique. For each "ACM-removal technique" combination, ATEM data were used to compute statistical indicators on short, fine and WHO asbestos fibers. Moreover, exposure was assessed taking into account the use of respiratory protective devices (RPD). As in previous studies, no simple relationship was found between results by PCOM and ATEM counting methods. Some ACM, such as asbestos-containing plasters, generated very high dust levels, and some techniques generated considerable levels of dust whatever the ACM treated. On the basis of these observations, recommendations were made to measure and control the occupational exposure limit. General prevention measures to be taken during work with ACM are also suggested. Finally, it is necessary to continue acquiring knowledge, in particular regarding RPD and the dust levels measured by ATEM for the activities not evaluated during this study.

Keggin-type polyoxometalate nanosheets: synthesis and characterization via scanning transmission electron microscopy.

PubMed

Hiyoshi, Norihito

2018-05-17

Polyoxometalate nanosheets were synthesized at the gas/liquid interface of an aqueous solution of Keggin-type silicotungstic acid, cesium chloride, and n-octylamine. The structure of the nanosheets was elucidated via aberration-corrected scanning transmission electron microscopy at the atomic and molecular levels.

Note on in situ (scanning) transmission electron microscopy study of liquid samples.

PubMed

Jiang, Nan

2017-08-01

Liquid cell (scanning) transmission electron microscopy has been developed rapidly, using amorphous SiN x membranes as electron transparent windows. The current interpretations of electron beam effects are mainly based on radiolytic processes. In this note, additional effects of the electric field due to electron-beam irradiation are discussed. The electric field can be produced by the charge accumulation due to the emission of secondary and Auger electrons. Besides various beam-induced phenomena, such as nanoparticle precipitation and gas bubble formation and motion, two other effects need to be considered; one is the change of Gibbs free energy of nucleation and the other is the violation of Brownian motion due to ion drifting driven by the electric field. Copyright © 2017 Elsevier B.V. All rights reserved.

Invited Review Article: Methods for imaging weak-phase objects in electron microscopy

PubMed Central

Glaeser, Robert M.

2013-01-01

Contrast has traditionally been produced in electron-microscopy of weak phase objects by simply defocusing the objective lens. There now is renewed interest, however, in using devices that apply a uniform quarter-wave phase shift to the scattered electrons relative to the unscattered beam, or that generate in-focus image contrast in some other way. Renewed activity in making an electron-optical equivalent of the familiar “phase-contrast” light microscope is based in part on the improved possibilities that are now available for device microfabrication. There is also a better understanding that it is important to take full advantage of contrast that can be had at low spatial frequency when imaging large, macromolecular objects. In addition, a number of conceptually new phase-plate designs have been proposed, thus increasing the number of options that are available for development. The advantages, disadvantages, and current status of each of these options is now compared and contrasted. Experimental results that are, indeed, superior to what can be accomplished with defocus-based phase contrast have been obtained recently with two different designs of phase-contrast aperture. Nevertheless, extensive work also has shown that fabrication of such devices is inconsistent, and that their working lifetime is short. The main limitation, in fact, appears to be electrostatic charging of any device that is placed into the electron diffraction pattern. The challenge in fabricating phase plates that are practical to use for routine work in electron microscopy thus may be more in the area of materials science than in the area of electron optics. PMID:24289381

Femtosecond few- to single-electron point-projection microscopy for nanoscale dynamic imaging

PubMed Central

Bainbridge, A. R.; Barlow Myers, C. W.; Bryan, W. A.

2016-01-01

Femtosecond electron microscopy produces real-space images of matter in a series of ultrafast snapshots. Pulses of electrons self-disperse under space-charge broadening, so without compression, the ideal operation mode is a single electron per pulse. Here, we demonstrate femtosecond single-electron point projection microscopy (fs-ePPM) in a laser-pump fs-e-probe configuration. The electrons have an energy of only 150 eV and take tens of picoseconds to propagate to the object under study. Nonetheless, we achieve a temporal resolution with a standard deviation of 114 fs (equivalent to a full-width at half-maximum of 269 ± 40 fs) combined with a spatial resolution of 100 nm, applied to a localized region of charge at the apex of a nanoscale metal tip induced by 30 fs 800 nm laser pulses at 50 kHz. These observations demonstrate real-space imaging of reversible processes, such as tracking charge distributions, is feasible whilst maintaining femtosecond resolution. Our findings could find application as a characterization method, which, depending on geometry, could resolve tens of femtoseconds and tens of nanometres. Dynamically imaging electric and magnetic fields and charge distributions on sub-micron length scales opens new avenues of ultrafast dynamics. Furthermore, through the use of active compression, such pulses are an ideal seed for few-femtosecond to attosecond imaging applications which will access sub-optical cycle processes in nanoplasmonics. PMID:27158637

Electronically tunable femtosecond all-fiber optical parametric oscillator for multi-photon microscopy

NASA Astrophysics Data System (ADS)

Hellwig, Tim; Brinkmann, Maximilian; Fallnich, Carsten

2018-02-01

We present a femtosecond fiber-based optical parametric oscillator (FOPO) for multiphoton microscopy with wavelength tuning by electronic repetition rate tuning in combination with a dispersive filter in the FOPO cavity. The all-spliced, all-fiber FOPO cavity is based on polarization-maintaining fibers and a broadband output coupler, allowing to get access to the resonant signal pulses as well as the idler pulses simultaneously. The system was pumped by a gain-switched fiber-coupled laser diode emitting pulses at a central wavelength of 1030 nm and an electronically tunable repetition frequency of about 2 MHz. The pump pulses were amplified in an Ytterbium fiber amplifier system with a pulse duration after amplification of 13 ps. Tuning of the idler (1140 nm - 1300 nm) and signal wavelengths (850 nm - 940 nm) was achieved by changing the repetition frequency of the pump laser by about 4 kHz. The generated signal pulses reached a pulse energy of up to 9.2 nJ at 920 nm and were spectrally broadened to about 6 nm in the FOPO by a combination of self-phase and cross-phase modulation. We showed external compression of the idler pulses at 920 nm to about 430 fs and appleid them to two-photon excitation microscopy with green fluorescent dyes. The presented system constitutes an important step towards a fully fiber-integrated all-electronically tunable and, thereby, programmable light source and already embodies a versatile and flexible light source for applications, e.g., for smart microscopy.

Microstructure of Mixed Surfactant Solutions by Electron Microscopy

NASA Astrophysics Data System (ADS)

Naranjo, Edward

1995-01-01

Surfactant mixtures add a new dimension to the design of complex fluid microstructure. By combining different surfactants it is not only possible to modify aggregate morphology and control the macrascopic properties of colloidal dispersions but also to produce a variety of novel synergistic phases. Mixed systems produce new microstructures by altering the intermolecular and interaggregate forces in ways impossible for single component systems. In this dissertation, we report on the phase behavior and microstructure of several synthetic and biological surfactant mixtures as elucidated by freeze-fracture and cryo-transmission electron microscopy. We have discovered that stable, spontaneous unilamellar vesicles can be prepared from aqueous mixtures of commercially available single-tailed cationic and anionic surfactants. Vesicle stability is determined by the length and volume of the hydrocarbon chains of the "catanionic" pairs. Mixtures containing bulky or branched surfactant pairs (C _{16}/C_{12 -14}) in water produce defect-free fairly monodisperse equilibrium vesicles at high dilution. In contrast, mixtures of catanionic surfactants with highly asymmetric tails (C_{16}/C_8 ) form phases of porous vesicles, dilute lamellar L_{alpha}, and anomalous isotropic L_3 phases. Images of the microstructure by freeze-fracture microscopy show that the L_3 phase consists of multiconnected self-avoiding bilayers with saddle shaped curvature. The forces between bilayers of vesicle-forming cationic and anionic surfactant mixtures were also measured using the Surface Force Apparatus (SFA). We find that the vesicles are stabilized by a long range electrostatic repulsion at large separations (>20 A) and an additional salt-independent repulsive force below 20 A. The measured forces correlate very well with the ternary phase diagram and the vesicle microstructures observed by electron microscopy. In addition to studying ionic surfactants, we have also done original work with

Static and Dynamic Electron Microscopy Investigations at the Atomic and Ultrafast Scales

NASA Astrophysics Data System (ADS)

Suri, Pranav Kumar

Advancements in the electron microscopy capabilities - aberration-corrected imaging, monochromatic spectroscopy, direct-electron detectors - have enabled routine visualization of atomic-scale processes with millisecond temporal resolutions in this decade. This, combined with progress in the transmission electron microscopy (TEM) specimen holder technology and nanofabrication techniques, allows comprehensive experiments on a wide range of materials in various phases via in situ methods. The development of ultrafast (sub-nanosecond) time-resolved TEM with ultrafast electron microscopy (UEM) has further pushed the envelope of in situ TEM to sub-nanosecond temporal resolution while maintaining sub-nanometer spatial resolution. A plethora of materials phenomena - including electron-phonon coupling, phonon transport, first-order phase transitions, bond rotation, plasmon dynamics, melting, and dopant atoms arrangement - are not yet clearly understood and could be benefitted with the current in situ TEM capabilities having atomic-level and ultrafast precision. Better understanding of these phenomena and intrinsic material dynamics (e.g. how phonons propagate in a material, what time-scales are involved in a first-order phase transition, how fast a material melts, where dopant atoms sit in a crystal) in new-generation and technologically important materials (e.g. two-dimensional layered materials, semiconductor and magnetic devices, rare-earth-element-free permanent magnets, unconventional superconductors) could bring a paradigm shift in their electronic, structural, magnetic, thermal and optical applications. Present research efforts, employing cutting-edge static and dynamic in situ electron microscopy resources at the University of Minnesota, are directed towards understanding the atomic-scale crystallographic structural transition and phonon transport in an iron-pnictide parent compound LaFeAsO, studying the mechanical stability of fast moving hard-drive heads in heat

The application of polyethylene glycol (PEG) to electron microscopy

PubMed Central

1980-01-01

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine- coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis. PMID:7400222

The application of polyethylene glycol (PEG) to electron microscopy.

PubMed

Wolosewick, J J

1980-08-01

The cytoplasm of cells from a variety of tissues has been viewed in sections (0.25-1 micrometers) devoid of any embedding resin. Glutaraldehyde- and osmium tetroxide-fixed tissues were infiltrated and embedded in a water-miscible wax, polyethylene glycol (PEG), and subsequently sectioned on dry glass or diamond knives. The PEG matrix was removed and the sections were placed on Formvarcarbon-polylysine-coated grids, dehydrated, dried by the critical-point method, and observed in either the high- or low-voltage electron microscope. Stereoscopic views of cells devoid of embedding resin present an image of cell utrastructure unobscured by electron-scattering resins similar to the image of whole, unembedded critical-point-dried or freeze-dried cultured cells observed by transmission electron microscopy. All organelles, including the cytoskeletal structures, are identified and appear not to have been damaged during processing, although membrane components appear somewhat less distinct. The absence of an embedding matrix eliminates the need for additional staining to increase contrast, unlike the situation with specimens embedded in standard electron-scattering resins. The PEG technique thus appears to be a valuable adjunct to conventional methods for ultrastructural analysis.

Micro-CT scouting for transmission electron microscopy of human tissue specimens

DOE Office of Scientific and Technical Information (OSTI.GOV)

Morales, A. G.; Stempinski, E. S.; XIAO, X.

Transmission electron microscopy (TEM) provides sub-nanometre-scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. Here, we describe micro-CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench-top micro-CT scanner with 10 m resolution was used to determine the location of patches of themore » mucous membrane in osmium-stained human nasal scraping samples. Furthermore, once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra-thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation. Lay description Electron microscopy provides very high levels of detail in a small area, and thus the question of where to look in an opaque sample, such as a stained tissue specimen, needs to be answered by sectioning the sample in small steps and examining the sections under a light microscope, until the region of interest is found. The search process can be lengthy and labor intensive, especially for a study involving a large number of samples. Small areas of interest can be missed in the process if not enough regions are examined. We also describe a method to directly locate the region of interest within a whole sample using micro-CT imaging, bypassing the need of blindly sectioning. Micro-CT enables locating the region within 3D space; this information provides a guide for sectioning the sample to expose that precise

Micro-CT scouting for transmission electron microscopy of human tissue specimens

DOE PAGES

Morales, A. G.; Stempinski, E. S.; XIAO, X.; ...

2016-02-08

Transmission electron microscopy (TEM) provides sub-nanometre-scale details in volumetric samples. Samples such as pathology tissue specimens are often stained with a metal element to enhance contrast, which makes them opaque to optical microscopes. As a result, it can be a lengthy procedure to find the region of interest inside a sample through sectioning. Here, we describe micro-CT scouting for TEM that allows noninvasive identification of regions of interest within a block sample to guide the sectioning step. In a tissue pathology study, a bench-top micro-CT scanner with 10 m resolution was used to determine the location of patches of themore » mucous membrane in osmium-stained human nasal scraping samples. Furthermore, once the regions of interest were located, the sample block was sectioned to expose that location, followed by ultra-thin sectioning and TEM to inspect the internal structure of the cilia of the membrane epithelial cells with nanometre resolution. This method substantially reduced the time and labour of the search process from typically 20 sections for light microscopy to three sections with no added sample preparation. Lay description Electron microscopy provides very high levels of detail in a small area, and thus the question of where to look in an opaque sample, such as a stained tissue specimen, needs to be answered by sectioning the sample in small steps and examining the sections under a light microscope, until the region of interest is found. The search process can be lengthy and labor intensive, especially for a study involving a large number of samples. Small areas of interest can be missed in the process if not enough regions are examined. We also describe a method to directly locate the region of interest within a whole sample using micro-CT imaging, bypassing the need of blindly sectioning. Micro-CT enables locating the region within 3D space; this information provides a guide for sectioning the sample to expose that precise

Effects of instrument imperfections on quantitative scanning transmission electron microscopy.

PubMed

Krause, Florian F; Schowalter, Marco; Grieb, Tim; Müller-Caspary, Knut; Mehrtens, Thorsten; Rosenauer, Andreas

2016-02-01

Several instrumental imperfections of transmission electron microscopes are characterized and their effects on the results of quantitative scanning electron microscopy (STEM) are investigated and quantified using simulations. Methods to either avoid influences of these imperfections during acquisition or to include them in reference calculations are proposed. Particularly, distortions inflicted on the diffraction pattern by an image-aberration corrector can cause severe errors of more than 20% if not accounted for. A procedure for their measurement is proposed here. Furthermore, afterglow phenomena and nonlinear behavior of the detector itself can lead to incorrect normalization of measured intensities. Single electrons accidentally impinging on the detector are another source of error but can also be exploited for threshold-less calibration of STEM images to absolute dose, incident beam current determination and measurement of the detector sensitivity. Copyright © 2015 Elsevier B.V. All rights reserved.

Alignment of large image series using cubic B-splines tessellation: application to transmission electron microscopy data.

PubMed

Dauguet, Julien; Bock, Davi; Reid, R Clay; Warfield, Simon K

2007-01-01

3D reconstruction from serial 2D microscopy images depends on non-linear alignment of serial sections. For some structures, such as the neuronal circuitry of the brain, very large images at very high resolution are necessary to permit reconstruction. These very large images prevent the direct use of classical registration methods. We propose in this work a method to deal with the non-linear alignment of arbitrarily large 2D images using the finite support properties of cubic B-splines. After initial affine alignment, each large image is split into a grid of smaller overlapping sub-images, which are individually registered using cubic B-splines transformations. Inside the overlapping regions between neighboring sub-images, the coefficients of the knots controlling the B-splines deformations are blended, to create a virtual large grid of knots for the whole image. The sub-images are resampled individually, using the new coefficients, and assembled together into a final large aligned image. We evaluated the method on a series of large transmission electron microscopy images and our results indicate significant improvements compared to both manual and affine alignment.

Revealing 3D Ultrastructure and Morphology of Stem Cell Spheroids by Electron Microscopy.

PubMed

Jaros, Josef; Petrov, Michal; Tesarova, Marketa; Hampl, Ales

2017-01-01

Cell culture methods have been developed in efforts to produce biologically relevant systems for developmental and disease modeling, and appropriate analytical tools are essential. Knowledge of ultrastructural characteristics represents the basis to reveal in situ the cellular morphology, cell-cell interactions, organelle distribution, niches in which cells reside, and many more. The traditional method for 3D visualization of ultrastructural components, serial sectioning using transmission electron microscopy (TEM), is very labor-intensive due to contentious TEM slice preparation and subsequent image processing of the whole collection. In this chapter, we present serial block-face scanning electron microscopy, together with complex methodology for spheroid formation, contrasting of cellular compartments, image processing, and 3D visualization. The described technique is effective for detailed morphological analysis of stem cell spheroids, organoids, as well as organotypic cell cultures.

Analytical electron microscopy of Mg-SiO smokes - A comparison with infrared and XRD studies

NASA Technical Reports Server (NTRS)

Rietmeijer, F. J. M.; Nuth, J. A.; Mackinnon, I. D. R.

1986-01-01

Analytical electron microscopy conducted for Mg-SiO smokes (experimentally obtained from samples previously characterized by IR spectroscopy) indicates that the microcrystallinity content of unannealed smokes increases with increased annealing for up to 30 hr. The growth of forsterite microcrystallites in the initially nonstoichiometric smokes may give rise to the contemporaneous growth of the SiO polymorph tridymite and MgO; after 4 hr of annealing, these react to form enstatite. It is suggested that XRD analysis and IR spectroscopy should be conducted in conjunction with detailed analytical electron microscopy for the detection of emerging crystallinity in vapor-phase condensates.

A graphene oxide-carbon nanotube grid for high-resolution transmission electron microscopy of nanomaterials.

PubMed

Zhang, Lina; Zhang, Haoxu; Zhou, Ruifeng; Chen, Zhuo; Li, Qunqing; Fan, Shoushan; Ge, Guanglu; Liu, Renxiao; Jiang, Kaili

2011-09-23

A novel grid for use in transmission electron microscopy is developed. The supporting film of the grid is composed of thin graphene oxide films overlying a super-aligned carbon nanotube network. The composite film combines the advantages of graphene oxide and carbon nanotube networks and has the following properties: it is ultra-thin, it has a large flat and smooth effective supporting area with a homogeneous amorphous appearance, high stability, and good conductivity. The graphene oxide-carbon nanotube grid has a distinct advantage when characterizing the fine structure of a mass of nanomaterials over conventional amorphous carbon grids. Clear high-resolution transmission electron microscopy images of various nanomaterials are obtained easily using the new grids.

Microstructural investigation of nickel silicide thin films and the silicide-silicon interface using transmission electron microscopy.

PubMed

Bhaskaran, M; Sriram, S; Mitchell, D R G; Short, K T; Holland, A S; Mitchell, A

2009-01-01

This article discusses the results of transmission electron microscopy (TEM)-based investigation of nickel silicide (NiSi) thin films grown on silicon. Nickel silicide is currently used as the CMOS technology standard for local interconnects and in electrical contacts. Films were characterized with a range of TEM-based techniques along with glancing angle X-ray diffraction. The nickel silicide thin films were formed by vacuum annealing thin films of nickel (50 nm) deposited on (100) silicon. The cross-sectional samples indicated a final silicide thickness of about 110 nm. This investigation studied and reports on three aspects of the thermally formed thin films: the uniformity in composition of the film using jump ratio maps; the nature of the interface using high resolution imaging; and the crystalline orientation of the thin films using selected-area electron diffraction (SAED). The analysis highlighted uniform composition in the thin films, which was also substantiated by spectroscopy techniques; an interface exhibiting the desired abrupt transition from silicide to silicon; and desired and preferential crystalline orientation corresponding to stoichiometric NiSi, supported by glancing angle X-ray diffraction results.

Morphometric, quantitative, and three-dimensional analysis of the heart muscle fibers of old rats: transmission electron microscopy and high-resolution scanning electron microscopy methods.

PubMed

Cury, Diego Pulzatto; Dias, Fernando José; Sosthenes, Marcia Consentino Kronka; Dos Santos Haemmerle, Carlos Alexandre; Ogawa, Koichi; Da Silva, Marcelo Cavenaghi Pereira; Mardegan Issa, João Paulo; Iyomasa, Mamie Mizusaki; Watanabe, Ii-Sei

2013-02-01

This research investigated the morphological, morphometric, and ultrastructural cardiomyocyte characteristics of male Wistar rats at 18 months of age. The animals were euthanized using an overdose of anesthesia (ketamine and xylazine, 150/10 mg/kg) and perfused transcardially, after which samples were collected for light microscopy, transmission electron microscopy, and high-resolution scanning electron microscopy. The results showed that cardiomyocyte arrangement was disposed parallel between the mitochondria and the A-, I-, and H-bands and their M- and Z-lines from the sarcomere. The sarcomere junction areas had intercalated disks, a specific structure of heart muscle. The ultrastructural analysis revealed several mitochondria of various sizes and shapes intermingled between the blood capillaries and their endothelial cells; some red cells inside vessels are noted. The muscle cell sarcolemma could be observed associated with the described structures. The cardiomyocytes of old rats presented an average sarcomere length of 2.071 ± 0.09 μm, a mitochondrial volume density (Vv) of 0.3383, a mitochondrial average area of 0.537 ± 0.278 μm(2), a mitochondrial average length of 1.024 ± 0.352 μm, an average mitochondrial cristae thickness of 0.038 ± 0.09 μm and a ratio of mitochondrial greater length/lesser length of 1.929 ± 0.965. Of the observed mitochondrial shapes, 23.4% were rounded, 45.3% were elongated, and 31.1% had irregular profiles. In this study, we analyzed the morphology and morphometry of cardiomyocytes in old rats, focusing on mitochondria. These data are important for researchers who focus the changes in cardiac tissue, especially changes owing to pathologies and drug administration that may or may not be correlated with aging. Copyright © 2012 Wiley Periodicals, Inc.

Improved Serial Sectioning Techniques for Correlative Light-Electron Microscopy Mapping of Human Langerhans Islets

PubMed Central

Saitoh, Sei; Ohno, Nobuhiko; Saitoh, Yurika; Terada, Nobuo; Shimo, Satoshi; Aida, Kaoru; Fujii, Hideki; Kobayashi, Tetsuro; Ohno, Shinichi

2018-01-01

Combined analysis of immunostaining for various biological molecules coupled with investigations of ultrastructural features of individual cells is a powerful approach for studies of cellular functions in normal and pathological conditions. However, weak antigenicity of tissues fixed by conventional methods poses a problem for immunoassays. This study introduces a method of correlative light and electron microscopy imaging of the same endocrine cells of compact and diffuse islets from human pancreatic tissue specimens. The method utilizes serial sections obtained from Epon-embedded specimens fixed with glutaraldehyde and osmium tetroxide. Double-immunofluorescence staining of thick Epon sections for endocrine hormones (insulin and glucagon) and regenerating islet-derived gene 1 α (REG1α) was performed following the removal of Epoxy resin with sodium ethoxide, antigen retrieval by autoclaving, and de-osmification treatment with hydrogen peroxide. The immunofluorescence images of endocrine cells were superimposed with the electron microscopy images of the same cells obtained from serial ultrathin sections. Immunofluorescence images showed well-preserved secretory granules in endocrine cells, whereas electron microscopy observations demonstrated corresponding secretory granules and intracellular organelles in the same cells. In conclusion, the correlative imaging approach developed by us may be useful for examining ultrastructural features in combination with immunolocalisation of endocrine hormones in the same human pancreatic islets. PMID:29622846

Metallocarbohedrenes: Transmission Electron Microscopy of Mass Gated Deposits

NASA Astrophysics Data System (ADS)

Castleman, M. E. Lyn, Jr.

2002-03-01

Titanium and zirconium Met-Car cluster ions have been detected from the direct laser vaporization of metal-graphite mixtures using time-of-flight mass spectrometry. Optimization of the production conditions enabled sufficient intensities to mass select and deposit Met-Cars on surfaces. High-resolution transmission electron microscopy images of mass gated Met-Car species reveals deposited nanocrystals 2 nm in diameter. Diffraction patterns indicate the presence of multiple species and shows that the deposits have spatial orientation. Lattice parameters have been extracted. The implication of the findings will be discussed. Support for the work has been from the AFOSR F49620-01-1-0122.

Further description of Cruzia tentaculata (Rudolphi, 1819) Travassos, 1917 (Nematoda: Cruzidae) by light and scanning electron microscopy.

PubMed

Adnet, F A O; Anjos, D H S; Menezes-Oliveira, A; Lanfredi, R M

2009-04-01

Species of Cruzia are parasites of the large intestine of marsupials, reptiles, amphibians, and mammalians. Cruzia tentaculata specimens were collected from the large intestine of Didelphis marsupialis (Mammalia: Didelphidae) from Colombia (new geographical record) and from Brazil and analyzed by light and scanning electron microscopy. The morphology of males and females by light microscopy corroborated most of the previous description and the ultrastructure by scanning electron microscopy evidence: the topography of the cuticle, deirids, amphids, phasmids in both sexes, a pair of papillae near the vulva opening, and the number and location of male caudal papillae, adding new features for species identification only observed by this technique.

Demonstration of correlative atomic force and transmission electron microscopy using actin cytoskeleton

PubMed Central

Yamada, Yutaro; Konno, Hiroki; Shimabukuro, Katsuya

2017-01-01

In this study, we present a new technique called correlative atomic force and transmission electron microscopy (correlative AFM/TEM) in which a targeted region of a sample can be observed under AFM and TEM. The ultimate goal of developing this new technique is to provide a technical platform to expand the fields of AFM application to complex biological systems such as cell extracts. Recent advances in the time resolution of AFM have enabled detailed observation of the dynamic nature of biomolecules. However, specifying molecular species, by AFM alone, remains a challenge. Here, we demonstrate correlative AFM/TEM, using actin filaments as a test sample, and further show that immuno-electron microscopy (immuno-EM), to specify molecules, can be integrated into this technique. Therefore, it is now possible to specify molecules, captured under AFM, by subsequent observation using immuno-EM. In conclusion, correlative AFM/TEM can be a versatile method to investigate complex biological systems at the molecular level. PMID:28828286

Molecular architecture of botulinum neurotoxin E revealed by single particle electron microscopy.

PubMed

Fischer, Audrey; Garcia-Rodriguez, Consuelo; Geren, Isin; Lou, Jianlong; Marks, James D; Nakagawa, Terunaga; Montal, Mauricio

2008-02-15

Clostridial botulinum neurotoxin (BoNT) causes a neuroparalytic condition recognized as botulism by arresting synaptic vesicle exocytosis. Although the crystal structures of full-length BoNT/A and BoNT/B holotoxins are known, the molecular architecture of the five other serotypes remains elusive. Here, we present the structures of BoNT/A and BoNT/E using single particle electron microscopy. Labeling of the particles with three different monoclonal antibodies raised against BoNT/E revealed the positions of their epitopes in the electron microscopy structure, thereby identifying the three hallmark domains of BoNT (protease, translocation, and receptor binding). Correspondingly, these antibodies selectively inhibit BoNT translocation activity as detected using a single molecule assay. The global structure of BoNT/E is strikingly different from that of BoNT/A despite strong sequence similarity. We postulate that the unique architecture of functionally conserved modules underlies the distinguishing attributes of BoNT/E and contributes to differences with BoNT/A.

4D imaging of transient structures and morphologies in ultrafast electron microscopy.

PubMed

Barwick, Brett; Park, Hyun Soon; Kwon, Oh-Hoon; Baskin, J Spencer; Zewail, Ahmed H

2008-11-21

With advances in spatial resolution reaching the atomic scale, two-dimensional (2D) and 3D imaging in electron microscopy has become an essential methodology in various fields of study. Here, we report 4D imaging, with in situ spatiotemporal resolutions, in ultrafast electron microscopy (UEM). The ability to capture selected-area-image dynamics with pixel resolution and to control the time separation between pulses for temporal cooling of the specimen made possible studies of fleeting structures and morphologies. We demonstrate the potential for applications with two examples, gold and graphite. For gold, after thermally induced stress, we determined the atomic structural expansion, the nonthermal lattice temperature, and the ultrafast transients of warping/bulging. In contrast, in graphite, striking coherent transients of the structure were observed in both image and diffraction, directly measuring, on the nanoscale, the longitudinal resonance period governed by Young's elastic modulus. The success of these studies demonstrates the promise of UEM in real-space imaging of dynamics.

Different patterns of collagen-proteoglycan interaction: a scanning electron microscopy and atomic force microscopy study.

PubMed

Raspanti, M; Congiu, T; Alessandrini, A; Gobbi, P; Ruggeri, A

2000-01-01

The extracellular matrix of unfixed, unstained rat corneal stroma, visualized with high-resolution scanning electron microscopy and atomic force microscopy after minimal preliminary treatment, appears composed of straight, parallel, uniform collagen fibrils regularly spaced by a three-dimensional, irregular network of thin, delicate proteoglycan filaments. Rat tail tendon, observed under identical conditions, appears instead made of heterogeneous, closely packed fibrils interwoven with orthogonal proteoglycan filaments. Pre-treatment with cupromeronic blue just thickens the filaments without affecting their spatial layout. Digestion with chondroitinase ABC rids the tendon matrix of all its interconnecting filaments while the corneal stroma architecture remains virtually unaffected, its fibrils always being separated by an evident interfibrillar spacing which is never observed in tendon. Our observations indicate that matrix proteoglycans are responsible for both the highly regular interfibrillar spacing which is distinctive of corneal stroma, and the strong interfibrillar binding observed in tendon. These opposite interaction patterns appear to be distinctive of different proteoglycan species. The molecular details of proteoglycan interactions are still incompletely understood and are the subject of ongoing research.

Double-shot MeV electron diffraction and microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Musumeci, P.; Cesar, D.; Maxson, J.

Here in this paper, we study by numerical simulations a time-resolved MeV electron scattering mode where two consecutive electron pulses are used to capture the evolution of a material sample on 10 ps time scales. The two electron pulses are generated by illuminating a photocathode in a radiofrequency photogun by two short laser pulses with adjustable delay. A streak camera/deflecting cavity is used after the sample to project the two electron bunches on two well separated regions of the detector screen. By using sufficiently short pulses, the 2D spatial information from each snapshot can be preserved. This “double-shot” technique enablesmore » the efficient capture of irreversible dynamics in both diffraction and imaging modes. Finally, in this work, we demonstrate both modes in start-to-end simulations of the UCLA Pegasus MeV microscope column.« less

Double-shot MeV electron diffraction and microscopy

DOE PAGES

Musumeci, P.; Cesar, D.; Maxson, J.

2017-05-19

Here in this paper, we study by numerical simulations a time-resolved MeV electron scattering mode where two consecutive electron pulses are used to capture the evolution of a material sample on 10 ps time scales. The two electron pulses are generated by illuminating a photocathode in a radiofrequency photogun by two short laser pulses with adjustable delay. A streak camera/deflecting cavity is used after the sample to project the two electron bunches on two well separated regions of the detector screen. By using sufficiently short pulses, the 2D spatial information from each snapshot can be preserved. This “double-shot” technique enablesmore » the efficient capture of irreversible dynamics in both diffraction and imaging modes. Finally, in this work, we demonstrate both modes in start-to-end simulations of the UCLA Pegasus MeV microscope column.« less

Helium ion microscopy and ultra-high-resolution scanning electron microscopy analysis of membrane-extracted cells reveals novel characteristics of the cytoskeleton of Giardia intestinalis.

PubMed

Gadelha, Ana Paula Rocha; Benchimol, Marlene; de Souza, Wanderley

2015-06-01

Giardia intestinalis presents a complex microtubular cytoskeleton formed by specialized structures, such as the adhesive disk, four pairs of flagella, the funis and the median body. The ultrastructural organization of the Giardia cytoskeleton has been analyzed using different microscopic techniques, including high-resolution scanning electron microscopy. Recent advances in scanning microscopy technology have opened a new venue for the characterization of cellular structures and include scanning probe microscopy techniques such as ultra-high-resolution scanning electron microscopy (UHRSEM) and helium ion microscopy (HIM). Here, we studied the organization of the cytoskeleton of G. intestinalis trophozoites using UHRSEM and HIM in membrane-extracted cells. The results revealed a number of new cytoskeletal elements associated with the lateral crest and the dorsal surface of the parasite. The fine structure of the banded collar was also observed. The marginal plates were seen linked to a network of filaments, which were continuous with filaments parallel to the main cell axis. Cytoplasmic filaments that supported the internal structures were seen by the first time. Using anti-actin antibody, we observed a labeling in these filamentous structures. Taken together, these data revealed new surface characteristics of the cytoskeleton of G. intestinalis and may contribute to an improved understanding of the structural organization of trophozoites. Copyright © 2015 Elsevier Inc. All rights reserved.

Schottky barrier height measurements of Cu/Si(001), Ag/Si(001), and Au/Si(001) interfaces utilizing ballistic electron emission microscopy and ballistic hole emission microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Balsano, Robert; Matsubayashi, Akitomo; LaBella, Vincent P., E-mail: vlabella@albany.edu

2013-11-15

The Schottky barrier heights of both n and p doped Cu/Si(001), Ag/Si(001), and Au/Si(001) diodes were measured using ballistic electron emission microscopy and ballistic hole emission microscopy (BHEM), respectively. Measurements using both forward and reverse ballistic electron emission microscopy (BEEM) and (BHEM) injection conditions were performed. The Schottky barrier heights were found by fitting to a linearization of the power law form of the Bell-Kaiser BEEM model. The sum of the n-type and p-type barrier heights are in good agreement with the band gap of silicon and independent of the metal utilized. The Schottky barrier heights are found to bemore » below the region of best fit for the power law form of the BK model, demonstrating its region of validity.« less

Immunogold scanning electron microscopy can reveal the polysaccharide architecture of xylem cell walls

PubMed Central

Sun, Yuliang; Juzenas, Kevin

2017-01-01

Abstract Immunofluorescence microscopy (IFM) and immunogold transmission electron microscopy (TEM) are the two main techniques commonly used to detect polysaccharides in plant cell walls. Both are important in localizing cell wall polysaccharides, but both have major limitations, such as low resolution in IFM and restricted sample size for immunogold TEM. In this study, we have developed a robust technique that combines immunocytochemistry with scanning electron microscopy (SEM) to study cell wall polysaccharide architecture in xylem cells at high resolution over large areas of sample. Using multiple cell wall monoclonal antibodies (mAbs), this immunogold SEM technique reliably localized groups of hemicellulosic and pectic polysaccharides in the cell walls of five different xylem structures (vessel elements, fibers, axial and ray parenchyma cells, and tyloses). This demonstrates its important advantages over the other two methods for studying cell wall polysaccharide composition and distribution in these structures. In addition, it can show the three-dimensional distribution of a polysaccharide group in the vessel lateral wall and the polysaccharide components in the cell wall of developing tyloses. This technique, therefore, should be valuable for understanding the cell wall polysaccharide composition, architecture and functions of diverse cell types. PMID:28398585

Electron microscopy study of microbial mat in the North Fiji basin hydrothermal vent

NASA Astrophysics Data System (ADS)

Park, H.; Kim, J. W.; Lee, J. W.

2017-12-01

Hydrothermal vent systems consisting of hydrothermal vent, hydrothermal sediment and microbial mat are widely spread around the ocean, particularly spreading axis, continental margin and back-arc basin. Scientists have perceived that the hydrothermal systems, which reflect the primeval earth environment, are one of the best places to reveal the origin of life and extensive biogeochemical process of microbe-mineral interaction. In the present study multiline of analytical methods (X-Ray Diffraction (XRD), Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM)) were utilized to investigate the mineralogy/chemistry of microbe-mineral interaction in hydrothermal microbial mat. Microbial mat samples were recovered by Canadian scientific submersible ROPOS on South Pacific North Fiji basin KIOST hydrothermal vent expedition 1602. XRD analysis showed that red-colored microbial mat contains Fe-oxides and Fe-oxyhydroxides. Various morphologies of minerals in the red-colored microbial mat observed by SEM are mainly showed sheath shaped, resembled with Leptothrix microbial structure, stalks shaped, similar with Marioprofundus microbial structure and globule shaped microbial structures. They are also detected with DNA analysis. The cross sectional observation of microbial structures encrusted with Fe-oxide and Fe-oxyhydroxide at a nano scale by Transmission Electron Microscopy (TEM) and Focused Ion Beam (FIB) technique was developed to verify the structural/biogeochemical properties in the microbe-mineral interaction. Systematic nano-scale measurements on the biomineralization in the microbial mat leads the understandings of biogeochemical environments around the hydrothermal vent.

Characterization of two-dimensional hexagonal boron nitride using scanning electron and scanning helium ion microscopy

NASA Astrophysics Data System (ADS)

Guo, Hongxuan; Gao, Jianhua; Ishida, Nobuyuki; Xu, Mingsheng; Fujita, Daisuke

2014-01-01

Characterization of the structural and physical properties of two-dimensional (2D) materials, such as layer number and inelastic mean free path measurements, is very important to optimize their synthesis and application. In this study, we characterize the layer number and morphology of hexagonal boron nitride (h-BN) nanosheets on a metallic substrate using field emission scanning electron microscopy (FE-SEM) and scanning helium ion microscopy (HIM). Using scanning beams of various energies, we could analyze the dependence of the intensities of secondary electrons on the thickness of the h-BN nanosheets. Based on the interaction between the scanning particles (electrons and helium ions) and h-BN nanosheets, we deduced an exponential relationship between the intensities of secondary electrons and number of layers of h-BN. With the attenuation factor of the exponential formula, we calculate the inelastic mean free path of electrons and helium ions in the h-BN nanosheets. Our results show that HIM is more sensitive and consistent than FE-SEM for characterizing the number of layers and morphology of 2D materials.

Advanced electron microscopy characterization of tri-layer rare-earth oxide superlattices

NASA Astrophysics Data System (ADS)

Phillips, Patrick; Disa, Ankit; Ismail-Beigi, Sohrab; Klie, Robert; University of Illinois-Chicago Team; Yale University Team

2015-03-01

Rare-earth nickelates are known to display complex electronic and magnetic behaviors owed to a very localized and sensitive Ni-site atomic and electronic structure. Toward realizing the goal of manipulating of the energetic ordering of Ni d orbitals and 2D conduction, the present work focuses on the experimental characterization of thin film superlattice structures consisting of alternating layers of LaTiO3 and LaNiO3 sandwiched between a dull insulator, LaAlO3. Using advanced scanning transmission electron microscopy (STEM)-based methods, properties such as interfacial sharpness, electron transfer, O presence, and local electronic structure can be probed at the atomic scale, and will be discussed at length. By combining both energy dispersive X-ray (EDX) and electronic energy loss (EEL) spectroscopies in an aberration-corrected STEM, it is possible to attain energy and spatial resolutions of 0.35 eV and 100 pm, respectively. Focus of the talk will remain not only on the aforementioned properties, but will also include details and parameters of the acquisitions to facilitate future characterization at this level.

Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry

DOE PAGES

Ophus, Colin; Ciston, Jim; Pierce, Jordan; ...

2016-02-29

The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, makingmore » it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Ultimately, simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals.« less

Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry

PubMed Central

Ophus, Colin; Ciston, Jim; Pierce, Jordan; Harvey, Tyler R.; Chess, Jordan; McMorran, Benjamin J.; Czarnik, Cory; Rose, Harald H.; Ercius, Peter

2016-01-01

The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, making it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals. PMID:26923483

Efficient linear phase contrast in scanning transmission electron microscopy with matched illumination and detector interferometry.

PubMed

Ophus, Colin; Ciston, Jim; Pierce, Jordan; Harvey, Tyler R; Chess, Jordan; McMorran, Benjamin J; Czarnik, Cory; Rose, Harald H; Ercius, Peter

2016-02-29

The ability to image light elements in soft matter at atomic resolution enables unprecedented insight into the structure and properties of molecular heterostructures and beam-sensitive nanomaterials. In this study, we introduce a scanning transmission electron microscopy technique combining a pre-specimen phase plate designed to produce a probe with structured phase with a high-speed direct electron detector to generate nearly linear contrast images with high efficiency. We demonstrate this method by using both experiment and simulation to simultaneously image the atomic-scale structure of weakly scattering amorphous carbon and strongly scattering gold nanoparticles. Our method demonstrates strong contrast for both materials, making it a promising candidate for structural determination of heterogeneous soft/hard matter samples even at low electron doses comparable to traditional phase-contrast transmission electron microscopy. Simulated images demonstrate the extension of this technique to the challenging problem of structural determination of biological material at the surface of inorganic crystals.

Ion Microscopy on Diamond

NASA Astrophysics Data System (ADS)

Manfredotti, Claudio

Because of its physical properties (strong radiation hardness, wide energy gap with a consequent extremely low dark current, very large electron and hole mobility) diamond is a very good candidate for nuclear particle detection, particularly in harsh environments or in conditions of strong radiation damage. Being commonly polycrystalline, diamond samples obtained by chemical vapour deposition (CVD) are not homogeneous, not only from the morphological point of view, but also from the electronic one. As a consequence, as it was indicated quite early starting from 1995, charge collection properties such as charge collection efficiency (cce) are not uniform, but they are depending on the site hit by incoming particle. Moreover, these properties are influenced by previous irradiations which are used in order to improve them and, finally, they are also dependent on the thickness of the sample, since the electronic non uniformity extends also in depth by affecting the profile of the electrical field from top to bottom electrode of the nuclear detector in the standard "sandwich" arrangement. By the use of focussed ion beams, it is possible to investigate these non uniformities by the aid of techniques like IBIC (Ion Beam Induced Charge) and IBIL (Ion Beam Induced Luminescence) with a space resolution of the order of 1 m. This relatively new kind of microscopy, which is called "ion microscopy", is capable not only to give 2D maps of cce, which can be quite precisely compared with morphological images obtained by Scanning Electron Microscopy (generally the grains display a much better cce than intergrain regions), but also to give the electric field profile from one electrode to the other one in a "lateral" arrangement of the ion beam. IBIL, by supplying 2D maps of luminescence intensity at different wavelength, can give information about the presence of specific radiative recombination centers and their distribution in the material. Finally, a new technique called XBIC (X

Preparation of high-quality ultrathin transmission electron microscopy specimens of a nanocrystalline metallic powder.

PubMed

Riedl, Thomas; Gemming, Thomas; Mickel, Christine; Eymann, Konrad; Kirchner, Alexander; Kieback, Bernd

2012-06-01

This article explores the achievable transmission electron microscopy specimen thickness and quality by using three different preparation methods in the case of a high-strength nanocrystalline Cu-Nb powder alloy. Low specimen thickness is essential for spatially resolved analyses of the grains in nanocrystalline materials. We have found that single-sided as well as double-sided low-angle Ar ion milling of the Cu-Nb powders embedded into epoxy resin produced wedge-shaped particles of very low thickness (<10 nm) near the edge. By means of a modified focused ion beam lift-out technique generating holes in the lamella interior large micrometer-sized electron-transparent regions were obtained. However, this lamella displayed a higher thickness at the rim of ≥30 nm. Limiting factors for the observed thicknesses are discussed including ion damage depths, backscattering, and surface roughness, which depend on ion type, energy, current density, and specimen motion. Finally, sections cut by ultramicrotomy at low stroke rate and low set thickness offered vast, several tens of square micrometers uniformly thin regions of ∼10-nm minimum thickness. As major drawbacks, we have detected a thin coating on the sections consisting of epoxy deployed as the embedding material and considerable nanoscale thickness variations. Copyright © 2011 Wiley Periodicals, Inc.

Secretory glands and microvascular systems imaged in aqueous solution by atmospheric scanning electron microscopy (ASEM).

PubMed

Yamazawa, Toshiko; Nakamura, Naotoshi; Sato, Mari; Sato, Chikara

2016-12-01

Exocrine glands, e.g., salivary and pancreatic glands, play an important role in digestive enzyme secretion, while endocrine glands, e.g., pancreatic islets, secrete hormones that regulate blood glucose levels. The dysfunction of these secretory organs immediately leads to various diseases, such as diabetes or Sjögren's syndrome, by poorly understood mechanisms. Gland-related diseases have been studied by optical microscopy (OM), and at higher resolution by transmission electron microscopy (TEM) of Epon embedded samples, which necessitates hydrophobic sample pretreatment. Here, we report the direct observation of tissue in aqueous solution by atmospheric scanning electron microscopy (ASEM). Salivary glands, lacrimal glands, and pancreas were fixed, sectioned into slabs, stained with phosphotungstic acid (PTA), and inspected in radical scavenger d-glucose solution from below by an inverted scanning electron microscopy (SEM), guided by optical microscopy from above to target the tissue substructures. A 2- to 3-µm specimen thickness was visualized by the SEM. In secretory cells, cytoplasmic vesicles and other organelles were clearly imaged at high resolution, and the former could be classified according to the degree of PTA staining. In islets of Langerhans, the microvascular system used as an outlet by the secretory cells was also clearly observed. Microvascular system is also critically involved in the onset of diabetic complications and was clearly visible in subcutaneous tissue imaged by ASEM. The results suggest the use of in-solution ASEM for histology and to study vesicle secretion systems. Further, the high-throughput of ASEM makes it a potential tool for the diagnosis of exocrine and endocrine-related diseases. © 2016 Wiley Periodicals, Inc.

A streaming multi-GPU implementation of image simulation algorithms for scanning transmission electron microscopy

DOE PAGES

Pryor, Alan; Ophus, Colin; Miao, Jianwei

2017-10-25

Simulation of atomic-resolution image formation in scanning transmission electron microscopy can require significant computation times using traditional methods. A recently developed method, termed plane-wave reciprocal-space interpolated scattering matrix (PRISM), demonstrates potential for significant acceleration of such simulations with negligible loss of accuracy. In this paper, we present a software package called Prismatic for parallelized simulation of image formation in scanning transmission electron microscopy (STEM) using both the PRISM and multislice methods. By distributing the workload between multiple CUDA-enabled GPUs and multicore processors, accelerations as high as 1000 × for PRISM and 15 × for multislice are achieved relative to traditionalmore » multislice implementations using a single 4-GPU machine. We demonstrate a potentially important application of Prismatic, using it to compute images for atomic electron tomography at sufficient speeds to include in the reconstruction pipeline. Prismatic is freely available both as an open-source CUDA/C++ package with a graphical user interface and as a Python package, PyPrismatic.« less

A streaming multi-GPU implementation of image simulation algorithms for scanning transmission electron microscopy.

PubMed

Pryor, Alan; Ophus, Colin; Miao, Jianwei

2017-01-01

Simulation of atomic-resolution image formation in scanning transmission electron microscopy can require significant computation times using traditional methods. A recently developed method, termed plane-wave reciprocal-space interpolated scattering matrix (PRISM), demonstrates potential for significant acceleration of such simulations with negligible loss of accuracy. Here, we present a software package called Prismatic for parallelized simulation of image formation in scanning transmission electron microscopy (STEM) using both the PRISM and multislice methods. By distributing the workload between multiple CUDA-enabled GPUs and multicore processors, accelerations as high as 1000 × for PRISM and 15 × for multislice are achieved relative to traditional multislice implementations using a single 4-GPU machine. We demonstrate a potentially important application of Prismatic , using it to compute images for atomic electron tomography at sufficient speeds to include in the reconstruction pipeline. Prismatic is freely available both as an open-source CUDA/C++ package with a graphical user interface and as a Python package, PyPrismatic .

Direct identification of metallic and semiconducting single-walled carbon nanotubes in scanning electron microscopy.

PubMed

Li, Jie; He, Yujun; Han, Yimo; Liu, Kai; Wang, Jiaping; Li, Qunqing; Fan, Shoushan; Jiang, Kaili

2012-08-08

Because of their excellent electrical and optical properties, carbon nanotubes have been regarded as extremely promising candidates for high-performance electronic and optoelectronic applications. However, effective and efficient distinction and separation of metallic and semiconducting single-walled carbon nanotubes are always challenges for their practical applications. Here we show that metallic and semiconducting single-walled carbon nanotubes on SiO(2) can have obviously different contrast in scanning electron microscopy due to their conductivity difference and thus can be effectively and efficiently identified. The correlation between conductivity and contrast difference has been confirmed by using voltage-contrast scanning electron microcopy, peak force tunneling atom force microscopy, and field effect transistor testing. This phenomenon can be understood via a proposed mechanism involving the e-beam-induced surface potential of insulators and the conductivity difference between metallic and semiconducting SWCNTs. This method demonstrates great promise to achieve rapid and large-scale distinguishing between metallic and semiconducting single-walled carbon nanotubes, adding a new function to conventional SEM.

A streaming multi-GPU implementation of image simulation algorithms for scanning transmission electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pryor, Alan; Ophus, Colin; Miao, Jianwei

Simulation of atomic-resolution image formation in scanning transmission electron microscopy can require significant computation times using traditional methods. A recently developed method, termed plane-wave reciprocal-space interpolated scattering matrix (PRISM), demonstrates potential for significant acceleration of such simulations with negligible loss of accuracy. In this paper, we present a software package called Prismatic for parallelized simulation of image formation in scanning transmission electron microscopy (STEM) using both the PRISM and multislice methods. By distributing the workload between multiple CUDA-enabled GPUs and multicore processors, accelerations as high as 1000 × for PRISM and 15 × for multislice are achieved relative to traditionalmore » multislice implementations using a single 4-GPU machine. We demonstrate a potentially important application of Prismatic, using it to compute images for atomic electron tomography at sufficient speeds to include in the reconstruction pipeline. Prismatic is freely available both as an open-source CUDA/C++ package with a graphical user interface and as a Python package, PyPrismatic.« less

Structural Studies of Amorphous Materials by Fluctuation Electron Microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Treacy, Michael M. J.

Fluctuation Electron Microscopy (FEM) is a technique that examines the fluctuations in electron scattering across a uniformly thin amorphous sample. The statistics of the intensity fluctuations, mean and variance, reveal any underlying medium-range order present in the structure. The goals of this project were: (1) To determine the fundamentals of the scattering physics that gives rise to the variance signal in fluctuation electron microscopy (FEM); (2) To use these discoveries to find ways to quantify FEM; (3) To apply the FEM method to interesting and technologically important families of amorphous materials, particularly those with important applications in energy-related processes. Excellent progress was made in items (1) and (2). In stage (3) we did not examine the metamict zircons, as proposed. Instead, we examined films of polycrystalline and amorphous semi-conducting diamond. Significant accomplishments are: (1) A Reverse Monte Carlo procedure was successfully implemented to invert FEM data into a structural model. This is computer-intensive, but it demonstrated that diffraction and FEM data from amorphous silicon are most consistent with a paracrystallite model. This means that there is more diamond-like topology present in amorphous silicon than is predicted by the continuous random network model. (2) There is significant displacement decoherence arising in diffraction from amorphous silicon and carbon. The samples are being bombarded by the electron beam and atoms do not stay still while being irradiated – much more than was formerly understood. The atom motions cause the destructive and constructive interferences in the diffraction pattern to fluctuate with time, and it is the time-averaged speckle that is being measured. The variance is reduced by a factor m, 4 ≤ m ≤ 1000, relative to that predicted by kinematical scattering theory. (3) Speckle intensity obeys a gamma distribution, where the mean intensitymore » $$ \\overline{I}\\ $$ and m are

Detection of atomic force microscopy cantilever displacement with a transmitted electron beam

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wagner, R.; Woehl, T. J.; Keller, R. R.

2016-07-25

The response time of an atomic force microscopy (AFM) cantilever can be decreased by reducing cantilever size; however, the fastest AFM cantilevers are currently nearing the smallest size that can be detected with the conventional optical lever approach. Here, we demonstrate an electron beam detection scheme for measuring AFM cantilever oscillations. The oscillating AFM tip is positioned perpendicular to and in the path of a stationary focused nanometer sized electron beam. As the tip oscillates, the thickness of the material under the electron beam changes, causing a fluctuation in the number of scattered transmitted electrons that are detected. We demonstratemore » detection of sub-nanometer vibration amplitudes with an electron beam, providing a pathway for dynamic AFM with cantilevers that are orders of magnitude smaller and faster than the current state of the art.« less

Atomic resolution elemental mapping using energy-filtered imaging scanning transmission electron microscopy with chromatic aberration correction.

PubMed

Krause, F F; Rosenauer, A; Barthel, J; Mayer, J; Urban, K; Dunin-Borkowski, R E; Brown, H G; Forbes, B D; Allen, L J

2017-10-01

This paper addresses a novel approach to atomic resolution elemental mapping, demonstrating a method that produces elemental maps with a similar resolution to the established method of electron energy-loss spectroscopy in scanning transmission electron microscopy. Dubbed energy-filtered imaging scanning transmission electron microscopy (EFISTEM) this mode of imaging is, by the quantum mechanical principle of reciprocity, equivalent to tilting the probe in energy-filtered transmission electron microscopy (EFTEM) through a cone and incoherently averaging the results. In this paper we present a proof-of-principle EFISTEM experimental study on strontium titanate. The present approach, made possible by chromatic aberration correction, has the advantage that it provides elemental maps which are immune to spatial incoherence in the electron source, coherent aberrations in the probe-forming lens and probe jitter. The veracity of the experiment is supported by quantum mechanical image simulations, which provide an insight into the image-forming process. Elemental maps obtained in EFTEM suffer from the effect known as preservation of elastic contrast, which, for example, can lead to a given atomic species appearing to be in atomic columns where it is not to be found. EFISTEM very substantially reduces the preservation of elastic contrast and yields images which show stability of contrast with changing thickness. The experimental application is demonstrated in a proof-of-principle study on strontium titanate. Copyright © 2017 Elsevier B.V. All rights reserved.

Scanning electron microscopy of Strongylus spp. in zebra.

PubMed

Els, H J; Malan, F S; Scialdo-Krecek, R C

1983-12-01

The external ultrastructure of the anterior and posterior extremities of the nematodes, Strongylus asini , Strongylus vulgaris, Strongylus equinus and Strongylus edentatus, was studied with scanning electron microscopy (SEM). Fresh specimens of S. asini were collected from the caecum, ventral colon and vena portae of Equus burchelli and Equus zebra hartmannae ; S. vulgaris from the caecum, colon and arteria ileocolica of E. burchelli ; S. equinus from the ventral colon of E. z. hartmannae and S. edentatus from the caecum and ventral colon of both zebras , during surveys of parasites in zebras in the Etosha Game Reserve, South West Africa/Namibia, and the Kruger National Park, Republic of South Africa. The worms were cleaned, fixed and mounted by standard methods and photographed in a JEOL JSM - 35C scanning electron microscope (SEM) operating at 12kV . The SEM showed the following differences: the tips of the external leaf-crowns varied and were fine and delicate in S. asini , coarse and broad in S. vulgaris and, in S. equinus and S. edentatus, closely adherent, separating into single elements for half their length. The excretory pores showed only slight variation, and the morphology of the copulatory bursae did not differ from those seen with light microscopy. The genital cones differed markedly: S. asini had a ventral triangular projection and laterally 2 finger-like projections: in S. vulgaris there were numerous bosses on the lateral and ventral aspects of the cone; in S. equinus 2 finger-like processes projected laterocaudally ; and in S. edentatus 2 pairs of papilla-like processes projected laterally on the ventral aspects, and a pair of rounded projections and a pair of hair-like structures adorned the dorsal aspects.(ABSTRACT TRUNCATED AT 250 WORDS)

Avoiding artefacts during electron microscopy of silver nanomaterials exposed to biological environments

PubMed Central

Goode, Angela E.; Skepper, Jeremy N.; Thorley, Andrew J.; Seiffert, Joanna M.; Chung, K. Fan; Tetley, Teresa D.; Shaffer, Milo S. P.; Ryan, Mary P.

2015-01-01

Electron microscopy has been applied widely to study the interaction of nanomaterials with proteins, cells and tissues at nanometre scale. Biological material is most commonly embedded in thermoset resins to make it compatible with the high vacuum in the electron microscope. Room temperature sample preparation protocols developed over decades provide contrast by staining cell organelles, and aim to preserve the native cell structure. However, the effect of these complex protocols on the nanomaterials in the system is seldom considered. Any artefacts generated during sample preparation may ultimately interfere with the accurate prediction of the stability and reactivity of the nanomaterials. As a case study, we review steps in the room temperature preparation of cells exposed to silver nanomaterials (AgNMs) for transmission electron microscopy imaging and analysis. In particular, embedding and staining protocols, which can alter the physicochemical properties of AgNMs and introduce artefacts thereby leading to a misinterpretation of silver bioreactivity, are scrutinised. Recommendations are given for the application of cryogenic sample preparation protocols, which simultaneously fix both particles and diffusible ions. By being aware of the advantages and limitations of different sample preparation methods, compromises or selection of different correlative techniques can be made to draw more accurate conclusions about the data. PMID:25606708

Staining of Tissue Sections for Electron Microscopy with Heavy Metals

PubMed Central

Watson, Michael L.

1958-01-01

Descriptions of three heavy metal stains and methods of application to tissue sections for electron microscopy are presented. Lead hydroxide stains rather selectively two types of particles in liver: those associated with the endoplasmic reticulum and containing ribonucleic acid and other somewhat larger particles. Barium hydroxide emphasizes certain bodies within vesicles of the Golgi region of hepatic cells. Alkalized lead acetate is useful as a general stain, as are also lead and barium hydroxides. PMID:13610936

Hepatic Subcellular Compartmentation of Cytoplasmic Phosphoenolpyruvate Carboxykinase Determined by Immunogold Electron Microscopy

NASA Astrophysics Data System (ADS)

Gao, Kuixiong; Cardell, Emma Lou; Morris, Randal E.; Giffin, Bruce F.; Cardell, Robert R.

1995-08-01

Phosphoenolpyruvate carboxykinase (PEPCK) is the rate-limiting gluconeogenic enzyme and in liver occurs in a lobular gradient from periportal to pericentral regions. The subcellular distribution of cytoplasmic PEPCK molecules within hepatocytes and its relationship to organelles have not been determined previously. In this study, we have used immunogold electron microscopy to evaluate the subcellar distribution of the enzyme, in addition to brightfield and epipolarized light microscopy. Cryosections (10 [mu]m) of perfusion-fixed rat liver were collected on silanated slides and immunostained using goat anti-rat PEPCK followed by 5-nm gold-labeled secondary and tertiary antibodies. Additionally, free-floating vibratome sections (25, 50, and 100 [mu]m) of perfusion-immersion-fixed rat liver were immunogold stained using goat anti-rat PEPCK and 5-nm gold-labeled secondary antibody, with and without silver enhancement. The immunogold labeled sections from both procedures were embedded in epoxy resin for the preparation of thin sections for electron microscopy. The results showed that the gold-labeled antibodies penetrated the entire thickness of cryosections, resulting in a high signal for PEPCK, but membranes in general, the smooth endoplasmic reticulum in particular, were not identifiable as electron dense unit membranes. On the other hand, the vibratome sections of well-fixed tissue allowed good visualization of the ultrastructure of cellular organelles, with the smooth endoplasmic reticulum appearing as vesicles and tubules with electron dense unit membranes; however, the penetration of the gold-labeled antibody was limited to cells at the surface of the vibratome sections. In both procedures, PEPCK, as indicated by gold particles, is predominantly in the glycogen areas of the cytosome and not in mitochondria, nuclei, Golgi apparatus, or other cell organelles. Hepatocytes in periportal regions have a compact subcellular distribution of PEPCK shown by gold particles

Investigating the use of in situ liquid cell scanning transmission electron microscopy

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nguy, Amanda

2016-02-19

Engineering nanoparticles with desired shape-dependent properties is the key to many applications in nanotechnology. Although many synthetic procedures exist to produce anisotropic gold nanoparticles, the dynamics of growth are typically unknown or hypothetical. In the case of seed-mediated growth in the presence of DNA into anisotropic nanoparticles, it is not known exactly how DNA directs growth into specific morphologies. A series of preliminary experiments were carried out to contribute to the investigation of the possible mechanism of DNA-mediated growth of gold nanoprisms into gold nanostars using liquid cell scanning transmission electron microscopy (STEM). Imaging in the liquid phase was achievedmore » through the use of a liquid cell platform and liquid cell holder that allow the sample to be contained within a “chip sandwich” between two electron transparent windows. Ex situ growth experiments were performed using Au-T30 NPrisms (30-base thymine oligonucleotide-coated gold nanoprisms) that are expected to grow into gold nanostars. Growth to form these nanostars were imaged using TEM (transmission electron microscopy) and liquid cell STEM (scanning transmission electron microscopy). An attempt to perform in situ growth experiments with the same Au-T30 nanoprisms revealed challenges in obtaining desired morphology results due to the environmental differences within the liquid cell compared to the ex situ environment. Different parameters in the experimental method were explored including fluid line set up, simultaneous and alternating reagent addition, and the effect of different liquid cell volumes to ensure adequate flow of reagents into the liquid cell. Lastly, the binding affinities were compared for T30 and A30 DNA incubated with gold nanoparticles using zeta potential measurements, absorption spectroscopy, and isothermal titration calorimetry (ITC). It was previously reported thymine bases have a lower binding affinity to gold surfaces than

Corneal collagen cross-linking: a confocal, electron, and light microscopy study of eye bank corneas.

PubMed

Dhaliwal, Jasmeet S; Kaufman, Stephen C

2009-01-01

The purpose of this study was to evaluate morphological changes induced by corneal collagen cross-linking in a human ex vivo cornea, using confocal, electron, and light microscopy. The central epithelium was partially removed from ex vivo human corneal buttons. Riboflavin 0.1% solution was applied before ultraviolet A light treatment and then for every 2 minutes for 30 minutes while the corneas were exposed to ultraviolet A light at a wavelength of 370 nm and intensity of 3 mW/cm(2). Each cornea was evaluated using confocal, electron, and light microscopy. Confocal microscopy demonstrated normal-appearing corneas on their initial pretreatment examination, with reduced stromal detail. After treatment, a superficial layer of highly reflective spherical structures (4-10 microm) was observed. Many of these hyperreflective structures appeared up to a depth of 300 microm. The remainder of the corneal stroma and endothelium appeared normal. Electron microscopy showed keratocyte apoptotic changes to a depth of 300 microm. No observable pathologic changes were seen on histology. Based on clinical studies, corneal cross-linking is a promising treatment that appears to be safe and to halt ectatic corneal disease progression. Initial European studies used animal models to extrapolate human protocols. In conjunction with clinical studies, we believe that human ex vivo corneal studies provide a means to evaluate the structural and morphological changes associated with this procedure, within human corneas, in a manner that cannot be accomplished in vivo.

Bright-field scanning confocal electron microscopy using a double aberration-corrected transmission electron microscope.

PubMed

Wang, Peng; Behan, Gavin; Kirkland, Angus I; Nellist, Peter D; Cosgriff, Eireann C; D'Alfonso, Adrian J; Morgan, Andrew J; Allen, Leslie J; Hashimoto, Ayako; Takeguchi, Masaki; Mitsuishi, Kazutaka; Shimojo, Masayuki

2011-06-01

Scanning confocal electron microscopy (SCEM) offers a mechanism for three-dimensional imaging of materials, which makes use of the reduced depth of field in an aberration-corrected transmission electron microscope. The simplest configuration of SCEM is the bright-field mode. In this paper we present experimental data and simulations showing the form of bright-field SCEM images. We show that the depth dependence of the three-dimensional image can be explained in terms of two-dimensional images formed in the detector plane. For a crystalline sample, this so-called probe image is shown to be similar to a conventional diffraction pattern. Experimental results and simulations show how the diffracted probes in this image are elongated in thicker crystals and the use of this elongation to estimate sample thickness is explored. Copyright © 2010 Elsevier B.V. All rights reserved.

Use of scanning electron microscopy and microanalysis to determine chloride content of concrete and raw materials.

DOT National Transportation Integrated Search

2013-02-01

Standard sample sets of cement and mortar formulations with known levels of Cl as well as concrete samples subject to Cl diffusion were all prepared for and analyzed with scanning electron microscopy (SEM) and electron microprobe (EPMA). Using x-ray ...

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy

PubMed Central

Rodríguez, José-Rodrigo; Turégano-López, Marta; DeFelipe, Javier; Merchán-Pérez, Angel

2018-01-01

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM). PMID:29568263

Neuroanatomy from Mesoscopic to Nanoscopic Scales: An Improved Method for the Observation of Semithin Sections by High-Resolution Scanning Electron Microscopy.

PubMed

Rodríguez, José-Rodrigo; Turégano-López, Marta; DeFelipe, Javier; Merchán-Pérez, Angel

2018-01-01

Semithin sections are commonly used to examine large areas of tissue with an optical microscope, in order to locate and trim the regions that will later be studied with the electron microscope. Ideally, the observation of semithin sections would be from mesoscopic to nanoscopic scales directly, instead of using light microscopy and then electron microscopy (EM). Here we propose a method that makes it possible to obtain high-resolution scanning EM images of large areas of the brain in the millimeter to nanometer range. Since our method is compatible with light microscopy, it is also feasible to generate hybrid light and electron microscopic maps. Additionally, the same tissue blocks that have been used to obtain semithin sections can later be used, if necessary, for transmission EM, or for focused ion beam milling and scanning electron microscopy (FIB-SEM).

Three-dimensional bright-field scanning transmission electron microscopy elucidate novel nanostructure in microbial biofilms.

PubMed

Hickey, William J; Shetty, Ameesha R; Massey, Randall J; Toso, Daniel B; Austin, Jotham

2017-01-01

Bacterial biofilms play key roles in environmental and biomedical processes, and understanding their activities requires comprehension of their nanoarchitectural characteristics. Electron microscopy (EM) is an essential tool for nanostructural analysis, but conventional EM methods are limited in that they either provide topographical information alone, or are suitable for imaging only relatively thin (<300 nm) sample volumes. For biofilm investigations, these are significant restrictions. Understanding structural relations between cells requires imaging of a sample volume sufficiently large to encompass multiple cells and the capture of both external and internal details of cell structure. An emerging EM technique with such capabilities is bright-field scanning transmission electron microscopy (BF-STEM) and in the present report BF-STEM was coupled with tomography to elucidate nanostructure in biofilms formed by the polycyclic aromatic hydrocarbon-degrading soil bacterium, Delftia acidovorans Cs1-4. Dual-axis BF-STEM enabled high-resolution 3-D tomographic recontructions (6-10 nm) visualization of thick (1250 and 1500 nm) sections. The 3-D data revealed that novel extracellular structures, termed nanopods, were polymorphic and formed complex networks within cell clusters. BF-STEM tomography enabled visualization of conduits formed by nanopods that could enable intercellular movement of outer membrane vesicles, and thereby enable direct communication between cells. This report is the first to document application of dual-axis BF-STEM tomography to obtain high-resolution 3-D images of novel nanostructures in bacterial biofilms. Future work with dual-axis BF-STEM tomography combined with correlative light electron microscopy may provide deeper insights into physiological functions associated with nanopods as well as other nanostructures. © 2016 The Authors Journal of Microscopy © 2016 Royal Microscopical Society.

Characterizing individual particles on tree leaves using computer automated scanning electron microscopy

Treesearch

D. L. Johnson; D. J. Nowak; V. A. Jouraeva

1999-01-01

Leaves from twenty-three deciduous tree species and five conifer species were collected within a limited geographic range (1 km radius) and evaluated for possible application of scanning electron microscopy and X-ray microanalysis techniques of individual particle analysis (IPA). The goal was to identify tree species with leaves suitable for the automated...

Imaging electronic states on topological semimetals using scanning tunneling microscopy

DOE PAGES

Gyenis, András; Inoue, Hiroyuki; Jeon, Sangjun; ...

2016-10-18

Following the intense studies on topological insulators, significant efforts have recently been devoted to the search for gapless topological systems. These materials not only broaden the topological classification of matter but also provide a condensed matter realization of various relativistic particles and phenomena previously discussed mainly in high energy physics. Weyl semimetals host massless, chiral, low-energy excitations in the bulk electronic band structure, whereas a symmetry protected pair of Weyl fermions gives rise to massless Dirac fermions.Weemployed scanning tunneling microscopy/spectroscopy to explore the behavior of electronic states both on the surface and in the bulk of topological semimetal phases. Bymore » mapping the quasiparticle interference (QPI) and emerging Landau levels at high magnetic field in Dirac semimetals Cd 3As 2 and Na 3Bi, we observed extended Dirac-like bulk electronic bands. QPI imaged on Weyl semimetal TaAs demonstrated the predicted momentum dependent delocalization of Fermi arc surface states in the vicinity of the surface projected Weyl nodes.« less

Visualization of carrier dynamics in p(n)-type GaAs by scanning ultrafast electron microscopy

PubMed Central

Cho, Jongweon; Hwang, Taek Yong; Zewail, Ahmed H.

2014-01-01

Four-dimensional scanning ultrafast electron microscopy is used to investigate doping- and carrier-concentration-dependent ultrafast carrier dynamics of the in situ cleaved single-crystalline GaAs(110) substrates. We observed marked changes in the measured time-resolved secondary electrons depending on the induced alterations in the electronic structure. The enhancement of secondary electrons at positive times, when the electron pulse follows the optical pulse, is primarily due to an energy gain involving the photoexcited charge carriers that are transiently populated in the conduction band and further promoted by the electron pulse, consistent with a band structure that is dependent on chemical doping and carrier concentration. When electrons undergo sufficient energy loss on their journey to the surface, dark contrast becomes dominant in the image. At negative times, however, when the electron pulse precedes the optical pulse (electron impact), the dynamical behavior of carriers manifests itself in a dark contrast which indicates the suppression of secondary electrons upon the arrival of the optical pulse. In this case, the loss of energy of material’s electrons is by collisions with the excited carriers. These results for carrier dynamics in GaAs(110) suggest strong carrier–carrier scatterings which are mirrored in the energy of material’s secondary electrons during their migration to the surface. The approach presented here provides a fundamental understanding of materials probed by four-dimensional scanning ultrafast electron microscopy, and offers possibilities for use of this imaging technique in the study of ultrafast charge carrier dynamics in heterogeneously patterned micro- and nanostructured material surfaces and interfaces. PMID:24469803

Visualization of carrier dynamics in p(n)-type GaAs by scanning ultrafast electron microscopy.

PubMed

Cho, Jongweon; Hwang, Taek Yong; Zewail, Ahmed H

2014-02-11

Four-dimensional scanning ultrafast electron microscopy is used to investigate doping- and carrier-concentration-dependent ultrafast carrier dynamics of the in situ cleaved single-crystalline GaAs(110) substrates. We observed marked changes in the measured time-resolved secondary electrons depending on the induced alterations in the electronic structure. The enhancement of secondary electrons at positive times, when the electron pulse follows the optical pulse, is primarily due to an energy gain involving the photoexcited charge carriers that are transiently populated in the conduction band and further promoted by the electron pulse, consistent with a band structure that is dependent on chemical doping and carrier concentration. When electrons undergo sufficient energy loss on their journey to the surface, dark contrast becomes dominant in the image. At negative times, however, when the electron pulse precedes the optical pulse (electron impact), the dynamical behavior of carriers manifests itself in a dark contrast which indicates the suppression of secondary electrons upon the arrival of the optical pulse. In this case, the loss of energy of material's electrons is by collisions with the excited carriers. These results for carrier dynamics in GaAs(110) suggest strong carrier-carrier scatterings which are mirrored in the energy of material's secondary electrons during their migration to the surface. The approach presented here provides a fundamental understanding of materials probed by four-dimensional scanning ultrafast electron microscopy, and offers possibilities for use of this imaging technique in the study of ultrafast charge carrier dynamics in heterogeneously patterned micro- and nanostructured material surfaces and interfaces.

Correlative fluorescence and scanning transmission electron microscopy of quantum dot-labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Bandmann, Vera; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy combined with scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, STEM can be accomplished in two ways. The microchip with the labeled cells and one microchip with a spacer are assembled into a special microfluidic device and imaged with dedicated high-voltage STEM. Alternatively, thin edges of cells can be studied with environmental scanning electron microscopy with a STEM detector, by placing a microchip with cells in a cooled wet environment. © 2014 Elsevier Inc. All rights reserved.

Energy-Filtered High-Resolution Electron Microscopy for Quantitative Solid State Structure Determination

DTIC Science & Technology

1997-01-01

is given by [19]: vP = e Ïne /(m0««0) , where e is the elementary charge, m0 is the electron rest mass, ne is the density of free electrons, and...and energy filtering systems. Fig. 8. EELS spectra acquired from a Ni/Ti multilayer specimen when the elctron beam is positioned at (a) Ti and (b) Ni...332 (1991). [11] T. Tanji, K. Urata, K. Ishizuka, Q. Ru, and A. Tonomura, Ultramicroscopy 49, 259–264 (1993). [12] D. E . Newbury, Microscopy: The Key

High-resolution electron microscopy and its applications.

PubMed

Li, F H

1987-12-01

A review of research on high-resolution electron microscopy (HREM) carried out at the Institute of Physics, the Chinese Academy of Sciences, is presented. Apart from the direct observation of crystal and quasicrystal defects for some alloys, oxides, minerals, etc., and the structure determination for some minute crystals, an approximate image-contrast theory named pseudo-weak-phase object approximation (PWPOA), which shows the image contrast change with crystal thickness, is described. Within the framework of PWPOA, the image contrast of lithium ions in the crystal of R-Li2Ti3O7 has been observed. The usefulness of diffraction analysis techniques such as the direct method and Patterson method in HREM is discussed. Image deconvolution and resolution enhancement for weak-phase objects by use of the direct method are illustrated. In addition, preliminary results of image restoration for thick crystals are given.

Platinum blue as an alternative to uranyl acetate for staining in transmission electron microscopy.

PubMed

Inaga, Sumire; Katsumoto, Tetsuo; Tanaka, Keiichi; Kameie, Toshio; Nakane, Hironobu; Naguro, Tomonori

2007-04-01

This paper introduces an aqueous solution of platinum blue (Pt-blue) as an alternative to uranyl acetate (UA) for staining in transmission electron microscopy (TEM). Pt-blue was prepared from a reaction of cis-dichlorodiamine-platinum (II) (cis-platin) with thymidine. When Pt-blue was dried on a microgrid and observed by TEM it showed a uniform appearance with tiny particles less than 1 nm in diameter. The effect of Pt-blue as an electron stain was then examined not only for positive staining of conventional ultrathin resin sections and counterstaining of post-embedding immuno-electron microscopy but also for negative staining. In ultrathin sections of the rat liver and renal glomerulus, Pt-blue provided good contrast images, especially in double staining combined with a lead stain (Pb). Almost all cell organelles were clearly observed with high contrast in these sections. Glycogen granules in the hepatic parenchymal cells were particularly electron dense in Pt-blue stained sections compared with those treated with UA. In longitudinal and transverse sections of budding influenza A viruses, a specific arrangement of rod-like structures, which correspond to the ribonucleoprotein complexes, was clearly shown in each virion stained with Pt-blue and Pb. When post-embedding immunoelectron microscopy was performed in ultrathin sections of HeLa cells embedded in Lowicryl K4M, the localization of Ki-67 protein was sufficiently detected even after Pt-blue and Pb staining. The present study also revealed that Pt-blue could be used for the negative staining of E. coli, allowing the visualization of a flagellum. These findings indicate that Pt-blue is a useful, safe, and easily obtainable electron stain that is an alternative to UA for TEM preparations.

Nanoscale deformation analysis with high-resolution transmission electron microscopy and digital image correlation

DOE PAGES

Wang, Xueju; Pan, Zhipeng; Fan, Feifei; ...

2015-09-10

We present an application of the digital image correlation (DIC) method to high-resolution transmission electron microscopy (HRTEM) images for nanoscale deformation analysis. The combination of DIC and HRTEM offers both the ultrahigh spatial resolution and high displacement detection sensitivity that are not possible with other microscope-based DIC techniques. We demonstrate the accuracy and utility of the HRTEM-DIC technique through displacement and strain analysis on amorphous silicon. Two types of error sources resulting from the transmission electron microscopy (TEM) image noise and electromagnetic-lens distortions are quantitatively investigated via rigid-body translation experiments. The local and global DIC approaches are applied for themore » analysis of diffusion- and reaction-induced deformation fields in electrochemically lithiated amorphous silicon. As a result, the DIC technique coupled with HRTEM provides a new avenue for the deformation analysis of materials at the nanometer length scales.« less

Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy.

PubMed

Peckys, Diana B; Baudoin, Jean-Pierre; Eder, Magdalena; Werner, Ulf; de Jonge, Niels

2013-01-01

Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the locations of individual EGFR dimer subunits. The sizes and distribution of dimers and higher order clusters of EGFRs were determined. The distance between labels bound to dimers amounted to 19 nm, consistent with a molecular model. A fraction of the EGFRs was found in higher order clusters with sizes ranging from 32-56 nm. ESEM can be used for quantitative whole cell screening studies of membrane receptors, and for the study of nanoparticle-cell interactions in general.

Epidermal growth factor receptor subunit locations determined in hydrated cells with environmental scanning electron microscopy

PubMed Central

Peckys, Diana B.; Baudoin, Jean-Pierre; Eder, Magdalena; Werner, Ulf; de Jonge, Niels

2013-01-01

Imaging single epidermal growth factor receptors (EGFR) in intact cells is presently limited by the available microscopy methods. Environmental scanning electron microscopy (ESEM) of whole cells in hydrated state in combination with specific labeling with gold nanoparticles was used to localize activated EGFRs in the plasma membranes of COS7 and A549 cells. The use of a scanning transmission electron microscopy (STEM) detector yielded a spatial resolution of 3 nm, sufficient to identify the locations of individual EGFR dimer subunits. The sizes and distribution of dimers and higher order clusters of EGFRs were determined. The distance between labels bound to dimers amounted to 19 nm, consistent with a molecular model. A fraction of the EGFRs was found in higher order clusters with sizes ranging from 32–56 nm. ESEM can be used for quantitative whole cell screening studies of membrane receptors, and for the study of nanoparticle-cell interactions in general. PMID:24022088

DESIGN NOTE: A modified Nanosurf scanning tunnelling microscope for ballistic electron emission microscopy and spectroscopy

NASA Astrophysics Data System (ADS)

Appelbaum, Ian; Thompson, Pete; van Schendel, P. J. A.

2006-04-01

We describe the design and implementation of modifications to an ambient STM with a slip stick approach mechanism to create a system capable of ballistic electron emission microscopy (BEEM) and spectroscopy (BEES). These modifications require building a custom sample holder which operates as a high gain transimpedance preamplifier. Results of microscopy and spectroscopy using a Au/n-GaAs Schottky device demonstrate the effectiveness of our design.

Using Graphene Liquid Cell Transmission Electron Microscopy to Study in Situ Nanocrystal Etching.

PubMed

Hauwiller, Matthew R; Ondry, Justin C; Alivisatos, A Paul

2018-05-17

Graphene liquid cell electron microscopy provides the ability to observe nanoscale chemical transformations and dynamics as the reactions are occurring in liquid environments. This manuscript describes the process for making graphene liquid cells through the example of graphene liquid cell transmission electron microscopy (TEM) experiments of gold nanocrystal etching. The protocol for making graphene liquid cells involves coating gold, holey-carbon TEM grids with chemical vapor deposition graphene and then using those graphene-coated grids to encapsulate liquid between two graphene surfaces. These pockets of liquid, with the nanomaterial of interest, are imaged in the electron microscope to see the dynamics of the nanoscale process, in this case the oxidative etching of gold nanorods. By controlling the electron beam dose rate, which modulates the etching species in the liquid cell, the underlying mechanisms of how atoms are removed from nanocrystals to form different facets and shapes can be better understood. Graphene liquid cell TEM has the advantages of high spatial resolution, compatibility with traditional TEM holders, and low start-up costs for research groups. Current limitations include delicate sample preparation, lack of flow capability, and reliance on electron beam-generated radiolysis products to induce reactions. With further development and control, graphene liquid cell may become a ubiquitous technique in nanomaterials and biology, and is already being used to study mechanisms governing growth, etching, and self-assembly processes of nanomaterials in liquid on the single particle level.

Microscopy and microanalysis 1996

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bailey, G.W.; Corbett, J.M.; Dimlich, R.V.W.

1996-12-31

The Proceedings of this Annual Meeting contain paper of members from the three societies. These proceedings emphasizes the common research interests and attempts to eliminate some unwanted overlap. Topics covered are: microscopic analysis of animals with altered gene expression and in-situ gene and antibody localizations, high-resolution elemental mapping of nucleoprofein interactions, plant biology and pathology, quantitative HREM analysis of perfect and defected materials, computational methods for TEM image analysis, high-resolution FESM in materials research, frontiers in polymer microscopy and microanalysis, oxidation and corrosion, micro XRD and XRF, molecular microspectroscopy and spectral imaging, advances in confocal and multidimensional light microscopy, analyticalmore » electron microscopy in biology, correlative microscopy in biological sciences, grain-boundary microengineering, surfaces and interfaces, telepresence microscopy in education and research, MSA educational outreach, quantitative electron probe microanalysis, frontiers of analytical electron microscopy, critical issues in ceramic microstructures, dynamic organization of the cell, pathology, microbiology, high-resolution biological and cryo SEM, and scanning-probe microscopy.« less

Balamuthia mandrillaris: Further morphological observations of trophozoites by light, scanning and transmission electron microscopy.

PubMed

González-Robles, Arturo; Lares-Villa, Fernando; Lares-Jiménez, Luis Fernando; Omaña-Molina, Maritza; Salazar-Villatoro, Lizbeth; Martínez-Palomo, Adolfo

2015-10-01

Additional morphological features of Balamuthia mandrillaris observed by light and electron microscopy are reported. Trophozoites were extremely pleomorphic: their cell shapes ranged from rounded to elongated and sometimes they appeared exceptionally stretched out and branched. By transmission electron microscopy it was possible to observe two different cytoplasmic areas, the ectoplasm and the endoplasm and often sections of rough endoplasmic reticulum were found in the transition zone. The cytoplasm was very fibrogranular and most of the organelles typically found in eukaryotic cells were observed. A particular finding was the presence of numerous mitochondria with a different structure from those of other free-living amoebae. The observations reported here may reinforce the morphological knowledge of this amoeba and provide a background for further analyses. Copyright © 2015 Elsevier Inc. All rights reserved.

Quantitative analysis of the effect of environmental-scanning electron microscopy on collagenous tissues.

PubMed

Lee, Woowon; Toussaint, Kimani C

2018-05-31

Environmental-scanning electron microscopy (ESEM) is routinely applied to various biological samples due to its ability to maintain a wet environment while imaging; moreover, the technique obviates the need for sample coating. However, there is limited research carried out on electron-beam (e-beam) induced tissue damage resulting from using the ESEM. In this paper, we use quantitative second-harmonic generation (SHG) microscopy to examine the effects of e-beam exposure from the ESEM on collagenous tissue samples prepared as either fixed, frozen, wet or dehydrated. Quantitative SHG analysis of tissues, before and after ESEM e-beam exposure in low-vacuum mode, reveals evidence of cross-linking of collagen fibers, however there are no structural differences observed in fixed tissue. Meanwhile wet-mode ESEM appears to radically alter the structure from a regular fibrous arrangement to a more random fiber orientation. We also confirm that ESEM images of collagenous tissues show higher spatial resolution compared to SHG microscopy, but the relative tradeoff with collagen specificity reduces its effectiveness in quantifying collagen fiber organization. Our work provides insight on both the limitations of the ESEM for tissue imaging, and the potential opportunity to use as a complementary technique when imaging fine features in the non-collagenous regions of tissue samples.

Trends in the Electron Microscopy Data Bank (EMDB)

PubMed Central

Patwardhan, Ardan

2017-01-01

Recent technological advances, such as the introduction of the direct electron detector, have transformed the field of cryo-EM and the landscape of molecular and cellular structural biology. This study analyses these trends from the vantage point of the Electron Microscopy Data Bank (EMDB), the public archive for three-dimensional EM reconstructions. Over 1000 entries were released in 2016, representing almost a quarter of the total number of entries (4431). Structures at better than 6 Å resolution now represent one of the fastest-growing categories, while the share of annually released tomography-related structures is approaching 20%. The use of direct electron detectors is growing very rapidly: they were used for 70% of the structures released in 2016, in contrast to none before 2011. Microscopes from FEI have an overwhelming lead in terms of usage, and the use of the RELION software package continues to grow rapidly after having attained a leading position in the field. China is rapidly emerging as a major player in the field, supplementing the US, Germany and the UK as the big four. Similarly, Tsinghua University ranks only second to the MRC Laboratory for Molecular Biology in terms of involvement in publications associated with cryo-EM structures at better than 4 Å resolution. Overall, the numbers point to a rapid democratization of the field, with more countries and institutes becoming involved. PMID:28580912

Trends in the Electron Microscopy Data Bank (EMDB).

PubMed

Patwardhan, Ardan

2017-06-01

Recent technological advances, such as the introduction of the direct electron detector, have transformed the field of cryo-EM and the landscape of molecular and cellular structural biology. This study analyses these trends from the vantage point of the Electron Microscopy Data Bank (EMDB), the public archive for three-dimensional EM reconstructions. Over 1000 entries were released in 2016, representing almost a quarter of the total number of entries (4431). Structures at better than 6 Å resolution now represent one of the fastest-growing categories, while the share of annually released tomography-related structures is approaching 20%. The use of direct electron detectors is growing very rapidly: they were used for 70% of the structures released in 2016, in contrast to none before 2011. Microscopes from FEI have an overwhelming lead in terms of usage, and the use of the RELION software package continues to grow rapidly after having attained a leading position in the field. China is rapidly emerging as a major player in the field, supplementing the US, Germany and the UK as the big four. Similarly, Tsinghua University ranks only second to the MRC Laboratory for Molecular Biology in terms of involvement in publications associated with cryo-EM structures at better than 4 Å resolution. Overall, the numbers point to a rapid democratization of the field, with more countries and institutes becoming involved.

On mapping subangstrom electron clouds with force microscopy.

PubMed

Wright, C Alan; Solares, Santiago D

2011-11-09

In 2004 Hembacher et al. (Science 2004, 305, 380-383) reported simultaneous higher-harmonics atomic force mocroscopy (AFM)/scanning tunneling microscopy (STM) images acquired while scanning a graphite surface with a tungsten tip. They interpreted the observed subatomic features in the AFM images as the signature of lobes of increased electron density at the tungsten tip apex. Although these intriguing images have stirred controversy, an in-depth theoretical feasibility study has not yet been produced. Here we report on the development of a method for simulating higher harmonics AFM images and its application to the same system. Our calculations suggest that four lobes of increased electron density are expected to be present at a W(001) tip apex atom and that the corresponding higher harmonics AFM images of graphite can exhibit 4-fold symmetry features. Despite these promising results, open questions remain since the calculated amplitudes of the higher harmonics generated by the short-range forces are on the order of hundredths of picometers, leading to very small corrugations in the theoretical images. Additionally, the complex, intermittent nature of the tip-sample interaction, which causes constant readjustment of the tip and sample orbitals as the tip approaches and retracts from the surface, prevents a direct quantitative connection between the electron density and the AFM image features.

Three-dimensional scanning transmission electron microscopy of biological specimens.

PubMed

de Jonge, Niels; Sougrat, Rachid; Northan, Brian M; Pennycook, Stephen J

2010-02-01

A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2-3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset.

Three-Dimensional Scanning Transmission Electron Microscopy of Biological Specimens

PubMed Central

de Jonge, Niels; Sougrat, Rachid; Northan, Brian M.; Pennycook, Stephen J.

2010-01-01

A three-dimensional (3D) reconstruction of the cytoskeleton and a clathrin-coated pit in mammalian cells has been achieved from a focal-series of images recorded in an aberration-corrected scanning transmission electron microscope (STEM). The specimen was a metallic replica of the biological structure comprising Pt nanoparticles 2–3 nm in diameter, with a high stability under electron beam radiation. The 3D dataset was processed by an automated deconvolution procedure. The lateral resolution was 1.1 nm, set by pixel size. Particles differing by only 10 nm in vertical position were identified as separate objects with greater than 20% dip in contrast between them. We refer to this value as the axial resolution of the deconvolution or reconstruction, the ability to recognize two objects, which were unresolved in the original dataset. The resolution of the reconstruction is comparable to that achieved by tilt-series transmission electron microscopy. However, the focal-series method does not require mechanical tilting and is therefore much faster. 3D STEM images were also recorded of the Golgi ribbon in conventional thin sections containing 3T3 cells with a comparable axial resolution in the deconvolved dataset. PMID:20082729

Microwave energy fixation for electron microscopy.

PubMed Central

Login, G. R.; Dvorak, A. M.

1985-01-01

We have demonstrated that microwave energy (MW) can be used in conjunction with chemical cross-linking agents in order to rapidly fix cell suspensions and tissue blocks for electron microscopy in 7-9 seconds. The optimal MW fixation method involved immersing tissues up to 1 cu cm in dilute aldehyde fixation and immediately irradiating the specimens in a conventional microwave oven for 9 seconds to 50 C. Ultrastructural preservation of samples irradiated by MW energy was comparable to that of the control samples immersed in aldehyde fixative for 2 hours at 25 C. Stereologic analysis showed that tissue blocks fixed by the MW fixation method did not cause organelles such as liver mitochondria and salivary gland granules to shrink or to swell. Potential applications for this new fixation technology include the investigation of rapid intracellular processes (eg, vesicular transport) and preservation of proteins that are difficult to demonstrate with routine fixation methods (eg, antigens and enzymes). Images Figure 4 Figure 5 Figure 2 Figure 3 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 PMID:3927740

High Dynamic Range Pixel Array Detector for Scanning Transmission Electron Microscopy.

PubMed

Tate, Mark W; Purohit, Prafull; Chamberlain, Darol; Nguyen, Kayla X; Hovden, Robert; Chang, Celesta S; Deb, Pratiti; Turgut, Emrah; Heron, John T; Schlom, Darrell G; Ralph, Daniel C; Fuchs, Gregory D; Shanks, Katherine S; Philipp, Hugh T; Muller, David A; Gruner, Sol M

2016-02-01

We describe a hybrid pixel array detector (electron microscope pixel array detector, or EMPAD) adapted for use in electron microscope applications, especially as a universal detector for scanning transmission electron microscopy. The 128×128 pixel detector consists of a 500 µm thick silicon diode array bump-bonded pixel-by-pixel to an application-specific integrated circuit. The in-pixel circuitry provides a 1,000,000:1 dynamic range within a single frame, allowing the direct electron beam to be imaged while still maintaining single electron sensitivity. A 1.1 kHz framing rate enables rapid data collection and minimizes sample drift distortions while scanning. By capturing the entire unsaturated diffraction pattern in scanning mode, one can simultaneously capture bright field, dark field, and phase contrast information, as well as being able to analyze the full scattering distribution, allowing true center of mass imaging. The scattering is recorded on an absolute scale, so that information such as local sample thickness can be directly determined. This paper describes the detector architecture, data acquisition system, and preliminary results from experiments with 80-200 keV electron beams.

Transmission Electron Microscopy of Cometary Residues from Micron-Sized Craters in the Stardust Al-Foils

NASA Technical Reports Server (NTRS)

Leroux, Hugues; Stroud, Rhonda M.; Dai, Zu Rong; Graham, Giles A.; Troadec, David; Bradley, John P.; Teslich, Nick; Borg, Janet; Kearsley, Anton T.; Horz, Friedrich

2008-01-01

We report Transmission Electron Microscopy (TEM) investigations of micro-craters that originated from hypervelocity impacts of comet 81P/Wild 2 dust particles on the aluminium foil of the Stardust collector. The craters were selected by Scanning Electron Microscopy (SEM) and then prepared by Focused Ion Beam (FIB) milling techniques in order to provide electron transparent cross-sections for TEM studies. The crater residues contain both amorphous and crystalline materials in varying proportions and compositions. The amorphous component is interpreted as resulting from shock melting during the impact and the crystalline phases as relict minerals. The latter show evidence for shock metamorphism. Based on the residue morphology and the compositional variation, the impacting particles are inferred to have been dominated by mixtures of submicron olivine, pyroxene and Fe-sulfide grains, in agreement with prior results of relatively coarse-grained mineral assemblages in the aerogel collector.

Retraction: Using the Medipix3 detector for direct electron imaging in the range 60 keV to 200 keV in electron microscopy Retraction: Using the Medipix3 detector for direct electron imaging in the range 60 keV to 200 keV in electron microscopy

NASA Astrophysics Data System (ADS)

Mir, J. A.; Plackett, R.; Shipsey, I.; dos Santos, J. M. F.

2018-01-01

The paper "Using the Medipix3 detector for direct electron imaging in the range 60keV to 200keV in electron microscopy" by J.A. Mir, R. Plackett, I. Shipsey and J.M.F. dos Santos has been retracted following the authors' request on the basis of the existence of a disagreement about the ownership of the data, to prevent conflict between collaborators.

A simple method for maintaining relative positions of separate tissue elements during processing for electron microscopy.

PubMed

Stirling, C A

1978-09-01

Molten (328 K) 20% gelatin is used as a 'glue' to hold together separate tissue elements or tissue elements that may be separated when cutting small blocks of tissue for plastic embedding. Standard aldehyde and osmium fixation, dehydration and epoxy embedding are compatible with this as is semi-thin sectioning for light microscopy or thin sectioning for electron microscopy.

The three dimensionality of cell membranes: lamellar to cubic membrane transition as investigated by electron microscopy.

PubMed

Chong, Ketpin; Deng, Yuru

2012-01-01

Biological membranes are generally perceived as phospholipid bilayer structures that delineate in a lamellar form the cell surface and intracellular organelles. However, much more complex and highly convoluted membrane organizations are ubiquitously present in many cell types under certain types of stress, states of disease, or in the course of viral infections. Their occurrence under pathological conditions make such three-dimensionally (3D) folded and highly ordered membranes attractive biomarkers. They have also stimulated great biomedical interest in understanding the molecular basis of their formation. Currently, the analysis of such membrane arrangements, which include tubulo-reticular structures (TRS) or cubic membranes of various subtypes, is restricted to electron microscopic methods, including tomography. Preservation of membrane structures during sample preparation is the key to understand their true 3D nature. This chapter discusses methods for appropriate sample preparations to successfully examine and analyze well-preserved highly ordered membranes by electron microscopy. Processing methods and analysis conditions for green algae (Zygnema sp.) and amoeba (Chaos carolinense), mammalian cells in culture and primary tissue cells are described. We also discuss methods to identify cubic membranes by transmission electron microscopy (TEM) with the aid of a direct template matching method and by computer simulation. A 3D analysis of cubic cell membrane topology by electron tomography is described as well as scanning electron microscopy (SEM) to investigate surface contours of isolated mitochondria with cubic membrane arrangement. Copyright © 2012 Elsevier Inc. All rights reserved.

EVALUATION OF COMPUTER-CONTROLLED SCANNING ELECTRON MICROSCOPY APPLIED TO AN AMBIENT URBAN AEROSOL SAMPLE

EPA Science Inventory

Concerns about the environmental and public health effects of particulate matter (PM) have stimulated interest in analytical techniques capable of measuring the size and chemical composition of individual aerosol particles. Computer-controlled scanning electron microscopy (CCSE...

Correlative fluorescence and electron microscopy of quantum dot labeled proteins on whole cells in liquid.

PubMed

Peckys, Diana B; Dukes, Madeline J; de Jonge, Niels

2014-01-01

Correlative fluorescence microscopy and scanning transmission electron microscopy (STEM) of cells fully immersed in liquid is a new methodology with many application areas. Proteins, in live cells immobilized on microchips, are labeled with fluorescent quantum dot (QD) nanoparticles. In this protocol, the epidermal growth factor receptor (EGFR) is labeled. The cells are fixed after a selected labeling time, for example, 5 min as needed to form EGFR dimers. The microchip with cells is then imaged with fluorescence microscopy. Thereafter, the microchip with the labeled cells and one with a spacer are assembled in a special microfluidic device and imaged with STEM.

Four-Dimensional Ultrafast Electron Microscopy: Insights into an Emerging Technique.

PubMed

Adhikari, Aniruddha; Eliason, Jeffrey K; Sun, Jingya; Bose, Riya; Flannigan, David J; Mohammed, Omar F

2017-01-11

Four-dimensional ultrafast electron microscopy (4D-UEM) is a novel analytical technique that aims to fulfill the long-held dream of researchers to investigate materials at extremely short spatial and temporal resolutions by integrating the excellent spatial resolution of electron microscopes with the temporal resolution of ultrafast femtosecond laser-based spectroscopy. The ingenious use of pulsed photoelectrons to probe surfaces and volumes of materials enables time-resolved snapshots of the dynamics to be captured in a way hitherto impossible by other conventional techniques. The flexibility of 4D-UEM lies in the fact that it can be used in both the scanning (S-UEM) and transmission (UEM) modes depending upon the type of electron microscope involved. While UEM can be employed to monitor elementary structural changes and phase transitions in samples using real-space mapping, diffraction, electron energy-loss spectroscopy, and tomography, S-UEM is well suited to map ultrafast dynamical events on materials surfaces in space and time. This review provides an overview of the unique features that distinguish these techniques and also illustrates the applications of both S-UEM and UEM to a multitude of problems relevant to materials science and chemistry.

Combined scanning transmission electron microscopy tilt- and focal series.

PubMed

Dahmen, Tim; Baudoin, Jean-Pierre; Lupini, Andrew R; Kübel, Christian; Slusallek, Philipp; de Jonge, Niels

2014-04-01

In this study, a combined tilt- and focal series is proposed as a new recording scheme for high-angle annular dark-field scanning transmission electron microscopy (STEM) tomography. Three-dimensional (3D) data were acquired by mechanically tilting the specimen, and recording a through-focal series at each tilt direction. The sample was a whole-mount macrophage cell with embedded gold nanoparticles. The tilt-focal algebraic reconstruction technique (TF-ART) is introduced as a new algorithm to reconstruct tomograms from such combined tilt- and focal series. The feasibility of TF-ART was demonstrated by 3D reconstruction of the experimental 3D data. The results were compared with a conventional STEM tilt series of a similar sample. The combined tilt- and focal series led to smaller "missing wedge" artifacts, and a higher axial resolution than obtained for the STEM tilt series, thus improving on one of the main issues of tilt series-based electron tomography.

Characterization of fast photoelectron packets in weak and strong laser fields in ultrafast electron microscopy.

PubMed

Plemmons, Dayne A; Tae Park, Sang; Zewail, Ahmed H; Flannigan, David J

2014-11-01

The development of ultrafast electron microscopy (UEM) and variants thereof (e.g., photon-induced near-field electron microscopy, PINEM) has made it possible to image atomic-scale dynamics on the femtosecond timescale. Accessing the femtosecond regime with UEM currently relies on the generation of photoelectrons with an ultrafast laser pulse and operation in a stroboscopic pump-probe fashion. With this approach, temporal resolution is limited mainly by the durations of the pump laser pulse and probe electron packet. The ability to accurately determine the duration of the electron packets, and thus the instrument response function, is critically important for interpretation of dynamics occurring near the temporal resolution limit, in addition to quantifying the effects of the imaging mode. Here, we describe a technique for in situ characterization of ultrashort electron packets that makes use of coupling with photons in the evanescent near-field of the specimen. We show that within the weakly-interacting (i.e., low laser fluence) regime, the zero-loss peak temporal cross-section is precisely the convolution of electron packet and photon pulse profiles. Beyond this regime, we outline the effects of non-linear processes and show that temporal cross-sections of high-order peaks explicitly reveal the electron packet profile, while use of the zero-loss peak becomes increasingly unreliable. Copyright © 2014 Elsevier B.V. All rights reserved.

Automated magnification calibration in transmission electron microscopy using Fourier analysis of replica images.

PubMed

van der Laak, Jeroen A W M; Dijkman, Henry B P M; Pahlplatz, Martin M M

2006-03-01

The magnification factor in transmission electron microscopy is not very precise, hampering for instance quantitative analysis of specimens. Calibration of the magnification is usually performed interactively using replica specimens, containing line or grating patterns with known spacing. In the present study, a procedure is described for automated magnification calibration using digital images of a line replica. This procedure is based on analysis of the power spectrum of Fourier transformed replica images, and is compared to interactive measurement in the same images. Images were used with magnification ranging from 1,000 x to 200,000 x. The automated procedure deviated on average 0.10% from interactive measurements. Especially for catalase replicas, the coefficient of variation of automated measurement was considerably smaller (average 0.28%) compared to that of interactive measurement (average 3.5%). In conclusion, calibration of the magnification in digital images from transmission electron microscopy may be performed automatically, using the procedure presented here, with high precision and accuracy.

Recent developments of the in situ wet cell technology for transmission electron microscopies.

PubMed

Chen, Xin; Li, Chang; Cao, Hongling

2015-03-21

In situ wet cells for transmission electron microscopy (TEM) and scanning transmission electron microscopy (STEM) allow studying structures and processes in a liquid environment with high temporal and spatial resolutions, and have been attracting increasing research interests in many fields. In this review, we highlight the structural and functional developments of the wet cells for TEM and STEM. One of the key features of the wet cells is the sealing technique used to isolate the liquid sample from the TEM/STEM vacuum environments, thus the existing in situ wet cells are grouped by different sealing methods. In this study, the advantages and shortcomings of each type of in situ wet cells are discussed, the functional developments of different wet cells are presented, and the future trends of the wet cell technology are addressed. It is suggested that in the future the in situ wet cell TEM/STEM technology will have an increasing impact on frontier nanoscale research.

Novel Solid Electrolytes for Li-Ion Batteries: A Perspective from Electron Microscopy Studies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ma, Cheng; Chi, Miaofang

2016-06-08

Solid electrolytes can simultaneously overcome two of the most formidable challenges of Li-ion batteries: the severe safety issues and insufficient energy densities. However, before they can be implemented in actual batteries, the ionic conductivity needs to be improved and the interface with electrodes must be optimized. The prerequisite for addressing these issues is a thorough understanding of the material’s behavior at the microscopic and/or the atomic level. (Scanning) transmission electron microscopy is a powerful tool for this purpose, as it can reach an ultrahigh spatial resolution. Here, we review recent electron microscopy investigations on the ion transport behavior in solidmore » electrolytes and their interfaces. Specifically, three aspects will be highlighted: the influence of grain interior atomic configuration on ionic conductivity, the contribution of grain boundaries, and the behavior of solid electrolyte/electrode interfaces. In conclusion, based on this, the perspectives for future research will be discussed.« less

TEMPy: a Python library for assessment of three-dimensional electron microscopy density fits.

PubMed

Farabella, Irene; Vasishtan, Daven; Joseph, Agnel Praveen; Pandurangan, Arun Prasad; Sahota, Harpal; Topf, Maya

2015-08-01

Three-dimensional electron microscopy is currently one of the most promising techniques used to study macromolecular assemblies. Rigid and flexible fitting of atomic models into density maps is often essential to gain further insights into the assemblies they represent. Currently, tools that facilitate the assessment of fitted atomic models and maps are needed. TEMPy (template and electron microscopy comparison using Python) is a toolkit designed for this purpose. The library includes a set of methods to assess density fits in intermediate-to-low resolution maps, both globally and locally. It also provides procedures for single-fit assessment, ensemble generation of fits, clustering, and multiple and consensus scoring, as well as plots and output files for visualization purposes to help the user in analysing rigid and flexible fits. The modular nature of TEMPy helps the integration of scoring and assessment of fits into large pipelines, making it a tool suitable for both novice and expert structural biologists.

Localized electronic structures of graphene oxide studied using scanning tunneling microscopy and spectroscopy.

PubMed

Katano, Satoshi; Wei, Tao; Sasajima, Takumi; Kasama, Ryuhei; Uehara, Yoichi

2018-06-21

We have used scanning tunneling microscopy (STM) to elucidate the nanoscale electronic structures of graphene oxide (GO). The unreduced GO layer was imaged using STM without reduction processes when deposited on a Au(111) surface covered with an octanethiolate self-assembled monolayer (C8S-SAM). The STM image of the GO sheet exhibits a grainy structure having a thickness of about 1 nm, which is in good agreement with the previous results obtained using atomic force microscopy (AFM). We found that the C8S-SAM suppresses the adsorption of water remaining on the substrate, which would be important to accomplish the nanoscale imaging of the unreduced GO by STM. Furthermore, we successfully detected the π and π* states localized in the GO sheet using scanning tunneling spectroscopy (STS). The π-π* gap energy and the gap center are not uniform within the GO sheet, indicating the existence of various sizes of the sp2 domain and evidence for the local electronic doping by the substituents.

SILVER IMPREGNATION OF ULTRATHIN SECTIONS FOR ELECTRON MICROSCOPY

PubMed Central

Marinozzi, Vittorio

1961-01-01

A new procedure is described for silver impregnation of thin sections for electron microscopy. Sections of various tissues, fixed in OsO4 and embedded in methacrylate, were treated with an ammoniacal silver solution, directly or after oxidation with periodic acid or hydrogen peroxide. After OsO4 fixation all cellular membranous systems exhibit a primary argentaffinity probably due to the reduction of ammoniacal silver solution by the reduced osmium bound to unsaturated lipids. Bleaching the sections with hydrogen peroxide removes the argentaffinity of protoplasmic structures. Treatment of the sections with periodic acid results in decreased argentaffinity of protoplasmic components while the argentaffinity of metaplasmic structures is greatly enhanced. The latter procedure appears particularly useful for enhancing the contrast of basement membranes. PMID:13766855

Neural plasticity explored by correlative two-photon and electron/SPIM microscopy

NASA Astrophysics Data System (ADS)

Allegra Mascaro, A. L.; Silvestri, L.; Costantini, I.; Sacconi, L.; Maco, B.; Knott, G. W.; Pavone, F. S.

2013-06-01

Plasticity of the central nervous system is a complex process which involves the remodeling of neuronal processes and synaptic contacts. However, a single imaging technique can reveal only a small part of this complex machinery. To obtain a more complete view, complementary approaches should be combined. Two-photon fluorescence microscopy, combined with multi-photon laser nanosurgery, allow following the real-time dynamics of single neuronal processes in the cerebral cortex of living mice. The structural rearrangement elicited by this highly confined paradigm of injury can be imaged in vivo first, and then the same neuron could be retrieved ex-vivo and characterized in terms of ultrastructural features of the damaged neuronal branch by means of electron microscopy. Afterwards, we describe a method to integrate data from in vivo two-photon fluorescence imaging and ex vivo light sheet microscopy, based on the use of major blood vessels as reference chart. We show how the apical dendritic arbor of a single cortical pyramidal neuron imaged in living mice can be found in the large-scale brain reconstruction obtained with light sheet microscopy. Starting from its apical portion, the whole pyramidal neuron can then be segmented and located in the correct cortical layer. With the correlative approach presented here, researchers will be able to place in a three-dimensional anatomic context the neurons whose dynamics have been observed with high detail in vivo.

Studies by immune electron microscopy of hepatitis B surface antigen in PLC/PRF/5 cells.

PubMed

Shibayama, T; Watanabe, T; Kojima, H; Yoshikawa, A; Watanabe, S; Kamimura, T; Suzuki, S; Ichida, F

1984-01-01

Electron microscopic studies of the morphology of hepatitis B surface antigen (HBsAg) produced by PLC/PRF/5 cells in vitro were carried out. Aggregates of 20-nm spherical particles in 3-day culture supernatants were observed by immune electron microscopy (IEM). Aggregates of tubular structures were found with IEM in the extracts of the cells. Tubular structures 18 to 22 nm in diameter were seen by electron microscopy (EM) in the cisternae of the endoplasmic reticulum in 2-3% of the cells. The tubular structures in the cytoplasm and extracts of PLC/PRF/5 cells resembled those observed in the hepatocytes of human carriers of hepatitis B virus (HBV). Intracellular localization of HBsAg in PLC/PRF/5 cells by direct peroxidase-conjugated antibody staining was observed on the tubular structures and the cisternal wall, which contained these structures. Rotation technique analysis indicated that the tubular structures were composed of 11 or 12 subunits.

Electron microscopy, histochemistry, and microbiology of bacterial adhesion in trichomycosis axillaris.

PubMed

Shelley, W B; Miller, M A

1984-06-01

Study of a case of trichomycosis axillaris by electron microscopy revealed a specific encapsulated Corynebacterium adhering to but not penetrating the hair shaft. External to this were two other biochemically distinctive pleomorphic Corynebacteria shown to be incapable of direct adherence to the hair. All three types were entrapped in a virtually insoluble cement substance, which they elaborate and which is responsible for the grossly visible colonization that is so characteristic of this disease.

Iplt--image processing library and toolkit for the electron microscopy community.

PubMed