Advanced electron microscopic techniques provide a deeper insight into the peculiar features of podocytes

2015 ◽  
Vol 309 (12) ◽  
pp. F1082-F1089 ◽  
Author(s):  
Tillmann Burghardt ◽  
Florian Hochapfel ◽  
Benjamin Salecker ◽  
Christine Meese ◽  
Hermann-Josef Gröne ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Electron Microscopic ◽  
Glomerular Filtration Barrier ◽  
Filtration Barrier ◽  
Future Work ◽  
Human And Mouse ◽  
Insight Into ◽  
Microscopic Techniques

Podocytes constitute the outer layer of the glomerular filtration barrier, where they form an intricate network of interdigitating foot processes which are connected by slit diaphragms. A hitherto unanswered puzzle concerns the question of whether slit diaphragms are established between foot processes of the same podocyte or between foot processes of different podocytes. By employing focused ion beam-scanning electron microscopy (FIB-SEM), we provide unequivocal evidence that slit diaphragms are formed between foot processes of different podocytes. We extended our investigations of the filtration slit by using dual-axis electron tomography of human and mouse podocytes as well as of Drosophila melanogaster nephrocytes. Using this technique, we not only find a single slit diaphragm which spans the filtration slit around the whole periphery of the foot processes but additional punctate filamentous contacts between adjacent foot processes. Future work will be necessary to determine the proteins constituting the two types of cell-cell contacts.

Advanced Physical Analysis Methodology for Yield and Reliability of 28-nm, Bulk-Si, Flip-Chip ICs Using SEM and Backside Deprocessing

2012 ◽  
Author(s):  
Yuanjing (Jane) Li ◽  
Steven Scott ◽  
Howard Lee Marks
Keyword(s):  
Flip Chip ◽  
Focused Ion Beam ◽  
Process Development ◽  
Ion Beam ◽  
Physical Analysis ◽  
Electron Microscopic ◽  
Analysis Methodology ◽  
Processing Techniques ◽  
20 Nm ◽  
Insight Into

Abstract This paper presents a backside chip-level physical analysis methodology using backside de-processing techniques in combination with optimized Scanning Electron Microscopic (SEM) imaging technique and Focused Ion Beam (FIB) cross sectioning to locate and analyze defects and faults in failing IC devices. The case studies illustrate the applications of the method for 28nm flip chip bulk Si CMOS devices and demonstrate how it is used in providing insight into the fab process and design for process and yield improvements. The methods are expected to play an even more important role during 20-nm process development and yield-ramping.

scholarly journals Volume electron microscopy: analyzing the lung

2020 ◽  
Author(s):  
Jan Philipp Schneider ◽  
Jan Hegermann ◽  
Christoph Wrede
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensional ◽  
Alveolar Epithelium ◽  
Practical Implementation ◽  
Electron Microscopic ◽  
Array Tomography ◽  
Three Dimensional Models

AbstractSince its entry into biomedical research in the first half of the twentieth century, electron microscopy has been a valuable tool for lung researchers to explore the lung’s delicate ultrastructure. Among others, it proved the existence of a continuous alveolar epithelium and demonstrated the surfactant lining layer. With the establishment of serial sectioning transmission electron microscopy, as the first “volume electron microscopic” technique, electron microscopy entered the third dimension and investigations of the lung’s three-dimensional ultrastructure became possible. Over the years, further techniques, ranging from electron tomography over serial block-face and focused ion beam scanning electron microscopy to array tomography became available. All techniques cover different volumes and resolutions, and, thus, different scientific questions. This review gives an overview of these techniques and their application in lung research, focusing on their fields of application and practical implementation. Furthermore, an introduction is given how the output raw data are processed and the final three-dimensional models can be generated.

scholarly journals Author response: Fully automated, sequential focused ion beam milling for cryo-electron tomography

2020 ◽  
Author(s):  
Tobias Zachs ◽  
Andreas Schertel ◽  
João Medeiros ◽  
Gregor L Weiss ◽  
Jannik Hugener ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Author Response ◽  
Cryo Electron Tomography ◽  
Focused Ion Beam Milling

scholarly journals Quantitative method for estimating stain density in electron microscopy of conventionally prepared biological specimens

2019 ◽  
Author(s):  
Andrea Fera ◽  
Qianping He ◽  
Guofeng Zhang ◽  
Richard D. Leapman
Keyword(s):  
Electron Microscopy ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Blood Platelets ◽  
Ion Beam ◽  
Simple Expression ◽  
Heavy Atoms ◽  
3D Electron Microscopy ◽  
Block Face ◽  
Microscopy Techniques

SummaryStain density is an important parameter for optimizing the quality of ultrastructural data obtained from several types of 3D electron microscopy techniques, including serial block-face electron microscopy (SBEM), and focused ion beam scanning electron microscopy (FIB-SEM). Here, we show how some straightforward measurements in the TEM can be used to determine the stain density based on a simple expression that we derive. Numbers of stain atoms per unit volume are determined from the measured ratio of the bright-field intensities from regions of the specimen that contain both pure embedding material and the embedded biological structures of interest. The determination only requires knowledge of the section thickness, which can either be estimated from the microtome setting, or from low-dose electron tomography, and the elastic scattering cross section for the heavy atoms used to stain the specimen. The method is tested on specimens of embedded blood platelets, brain tissue, and liver tissue.

scholarly journals Automated cryo-lamella preparation for high-throughput in-situ structural biology

2019 ◽  
Author(s):  
Genevieve Buckley ◽  
Gediminas Gervinskas ◽  
Cyntia Taveneau ◽  
Hari Venugopal ◽  
James C. Whisstock ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Structural Biology ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Automated Method ◽  
Cellular Environment ◽  
Transmission Electron

AbstractCryo-transmission electron tomography (cryo-ET) in association with cryo-focused ion beam (cryo-FIB) milling enables structural biology studies to be performed directly within the cellular environment. Cryo-preserved cells are milled and a lamella with a thickness of 200-300 nm provides an electron transparent window suitable for cryo-ET imaging. Cryo-FIB milling is an effective method, but it is a tedious and time-consuming process, which typically results in ~10 lamellae per day. Here, we introduce an automated method to reproducibly prepare cryo-lamellae on a grid and reduce the amount of human supervision. We tested the routine on cryo-preserved Saccharomyces cerevisiae and demonstrate that this method allows an increased throughput, achieving a rate of 5 lamellae/hour without the need to supervise the FIB milling. We demonstrate that the quality of the lamellae is consistent throughout the preparation and their compatibility with cryo-ET analyses.

scholarly journals A modular platform for automated cryo-FIB workflows

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Sven Klumpe ◽  
Herman K H Fung ◽  
Sara K Goetz ◽  
Ievgeniia Zagoriy ◽  
Bernhard Hampoelz ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Structural Integrity ◽  
Electron Tomography ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Software Tool ◽  
3 Dimensional ◽  
Improved Stability ◽  
Modular Platform ◽  
Beam Currents

Lamella micromachining by focused ion beam milling at cryogenic temperature (cryo-FIB) has matured into a preparation method widely used for cellular cryo-electron tomography. Due to the limited ablation rates of low Ga+ ion beam currents required to maintain the structural integrity of vitreous specimens, common preparation protocols are time-consuming and labor intensive. The improved stability of new generation cryo-FIB instruments now enables automated operations. Here, we present an open-source software tool, SerialFIB, for creating automated and customizable cryo-FIB preparation protocols. The software encompasses a graphical user interface for easy execution of routine lamellae preparations, a scripting module compatible with available Python packages, and interfaces with 3-dimensional correlative light and electron microscopy (CLEM) tools. SerialFIB enables the streamlining of advanced cryo-FIB protocols such as multi-modal imaging, CLEM-guided lamella preparation and in situ lamella lift-out procedures. Our software therefore provides a foundation for further development of advanced cryogenic imaging and sample preparation protocols.

Point Defect Clusters and Dislocations in FIB Irradiated Nanocrystalline Aluminum Films: An Electron Tomography and Aberration-Corrected High-Resolution ADF-STEM Study

2011 ◽  
Vol 17 (6) ◽  
pp. 983-990 ◽  
Author(s):  
Hosni Idrissi ◽  
Stuart Turner ◽  
Masatoshi Mitsuhara ◽  
Binjie Wang ◽  
Satoshi Hata ◽  
...  
Keyword(s):  
High Resolution ◽  
Point Defect ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Dark Field ◽  
Internal Stresses ◽  
Defect Clusters ◽  
Point Defect Clusters ◽  
Aberration Corrected

AbstractFocused ion beam (FIB) induced damage in nanocrystalline Al thin films has been characterized using advanced transmission electron microscopy techniques. Electron tomography was used to analyze the three-dimensional distribution of point defect clusters induced by FIB milling, as well as their interaction with preexisting dislocations generated by internal stresses in the Al films. The atomic structure of interstitial Frank loops induced by irradiation, as well as the core structure of Frank dislocations, has been resolved with aberration-corrected high-resolution annular dark-field scanning TEM. The combination of both techniques constitutes a powerful tool for the study of the intrinsic structural properties of point defect clusters as well as the interaction of these defects with preexisting or deformation dislocations in irradiated bulk or nanostructured materials.

scholarly journals Insight into <i>Emiliania huxleyi</i> coccospheres by focused ion beam sectioning

2015 ◽  
Vol 12 (3) ◽  
pp. 825-834 ◽  
Author(s):  
R. Hoffmann ◽  
C. Kirchlechner ◽  
G. Langer ◽  
A. S. Wochnik ◽  
E. Griesshaber ◽  
...  
Keyword(s):  
Electron Micrographs ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Emiliania Huxleyi ◽  
Exponential Growth Phase ◽  
Cell Diameter ◽  
Scanning Electron Micrographs ◽  
Ongoing Debate ◽  
Insight Into ◽  
Sinking Velocity

Abstract. Coccospheres of a cultured Emiliania huxleyi clone were sampled in the exponential growth phase and sectioned using a focused ion beam microscope. An average of 69 sections and the corresponding secondary electron micrographs per coccosphere provided detailed information on coccosphere architecture. The coccospheres feature 2–3 layers on average and 20 coccoliths per cell, of which only 15 can be seen in conventional scanning electron micrographs. The outer coccosphere diameter was positively correlated with the number of coccolith layers. By contrast, the inner coccosphere diameter (around 4.36 μm), and hence the cell diameter, was quasi-constant. Coccoliths were not evenly distributed across the coccosphere, resulting more often than not in one part of the coccosphere displaying more coccolith layers than the other. The architectural data allowed for the calculation of the PIC $/$ POC ratio, the density and the sinking velocity of individual cells. The correlation of these parameters has implications for the ongoing debate on the function of coccoliths.

scholarly journals The structure of the COPI coat determined within the cell

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Yury S Bykov ◽  
Miroslava Schaffer ◽  
Svetlana O Dodonova ◽  
Sahradha Albert ◽  
Jürgen M Plitzko ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Structural Model ◽  
Electron Tomography ◽  
Ion Beam ◽  
Membrane Thickness ◽  
In Vitro Reconstitution ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

COPI-coated vesicles mediate trafficking within the Golgi apparatus and from the Golgi to the endoplasmic reticulum. The structures of membrane protein coats, including COPI, have been extensively studied with in vitro reconstitution systems using purified components. Previously we have determined a complete structural model of the in vitro reconstituted COPI coat (Dodonova et al., 2017). Here, we applied cryo-focused ion beam milling, cryo-electron tomography and subtomogram averaging to determine the native structure of the COPI coat within vitrified Chlamydomonas reinhardtii cells. The native algal structure resembles the in vitro mammalian structure, but additionally reveals cargo bound beneath β’–COP. We find that all coat components disassemble simultaneously and relatively rapidly after budding. Structural analysis in situ, maintaining Golgi topology, shows that vesicles change their size, membrane thickness, and cargo content as they progress from cis to trans, but the structure of the coat machinery remains constant.

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