scholarly journals In-situ fiducial markers for 3D correlative cryo- fluorescence and FIB-SEM imaging

2021 ◽  
Author(s):  
Nadav Scher ◽  
Katya Rechav ◽  
Perrine Paul-Gilloteaux ◽  
Ori Avinoam
Keyword(s):  
Lipid Droplets ◽  
3D Imaging ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Fiducial Markers ◽  
Dual Beam ◽  
Sem Imaging ◽  
Cryogenic Conditions

Imaging of cells and tissues has improved significantly over the last decade. Dual-beam instruments with a focused ion beam mounted on a scanning electron microscope (FIB-SEM), which offer high-resolution 3D imaging of large volumes and fields-of-view are becoming widely used in the life sciences. FIB-SEM has most recently been implemented on fully hydrated, cryo-immobilized, biological samples. However, correlative light and electron microscopy (CLEM) workflows combining cryo- fluorescence microscopy (cryo-FM) and FIB-SEM are not yet commonly available. Here, we demonstrate that fluorescently labeled lipid droplets can serve as in-situ fiducial markers for correlating cryo- FM and FIB-SEM datasets, and that this approach can be used to target the acquisition of large FIB-SEM stacks spanning tens of microns under cryogenic conditions. We also show that cryo-FIB-SEM imaging is particularly informative for questions related to organelle structure and inter-organellar contacts, nuclear organization and mineral deposits in cells.

scholarly journals Application of FIB/SEM and TEM to Bit Failure Analyses in SRAM Arrays

2003 ◽  
Vol 782 ◽  
Author(s):  
Wentao Qin ◽  
Alex Volinsky ◽  
Larry Rice ◽  
Lorraine Johnston ◽  
David Theodore
Keyword(s):  
Focused Ion Beam ◽  
Ion Beam ◽  
Embedded Memory ◽  
Area Of Interest ◽  
Microelectronic Devices ◽  
Dual Beam ◽  
Sem Imaging ◽  
Lift Off ◽  
Failure Site

ABSTRACTMany microelectronic chips contain embedded memory arrays. A single SRAM bit-cell contains several transistors. Failure of any of the transistors makes the entire bit-cell inoperable. Dual-beam Focused Ion Beam (FIB) combines the slicing capability of FIB with in-situ SEM imaging. The combination offers unparalleled precision in looking for root causes of failures in microelectronic devices. Once a failure site is located, an FIB lift-off method can be used to prepare a TEM sample containing the area of interest. Further structural, elemental information can then be acquired from the failure site. We report here analyses of single and multiple bit failures in SRAM arrays carried out using FIB/SEM, and in two cases TEM imaging and EDS/PEELS. Root causes of bit failures including remnant seed-layer metal between stacked vias have been identified.

To Eliminate Curtain Effect of FIB TEM Samples by a Combination of Sample Dicing and Backside Milling

2014 ◽  
Author(s):  
Jian-Shing Luo ◽  
Hsiu Ting Lee
Keyword(s):  
Sample Preparation ◽  
Focused Ion Beam ◽  
Preparation Method ◽  
Low Cost ◽  
Ion Beam ◽  
Sample Preparation Method ◽  
Site Specific ◽  
Dual Beam ◽  
Transmission Electron

Abstract Several methods are used to invert samples 180 deg in a dual beam focused ion beam (FIB) system for backside milling by a specific in-situ lift out system or stages. However, most of those methods occupied too much time on FIB systems or requires a specific in-situ lift out system. This paper provides a novel transmission electron microscopy (TEM) sample preparation method to eliminate the curtain effect completely by a combination of backside milling and sample dicing with low cost and less FIB time. The procedures of the TEM pre-thinned sample preparation method using a combination of sample dicing and backside milling are described step by step. From the analysis results, the method has applied successfully to eliminate the curtain effect of dual beam FIB TEM samples for both random and site specific addresses.

In-Situ Dual Beam (FIBSEM) Techniques for Probe Pad Deposition and Dielectric Integrity Inspection in 0.2 μm Technology DRAM Single Cells

1999 ◽  
Author(s):  
Gunnar Zimmermann ◽  
Richard Chapman
Keyword(s):  
Electron Beam ◽  
Single Cell ◽  
Single Cells ◽  
Ion Beam ◽  
Gate Oxide ◽  
Ion Milling ◽  
Dual Beam ◽  
Sem Imaging ◽  
Innovative Techniques

Abstract Dual beam FIBSEM systems invite the use of innovative techniques to localize IC fails both electrically and physically. For electrical localization, we present a quick and reliable in-situ FIBSEM technique to deposit probe pads with very low parasitic leakage (Ipara < 4E-11A at 3V). The probe pads were Pt, deposited with ion beam assistance, on top of highly insulating SiOx, deposited with electron beam assistance. The buried plate (n-Band), p-well, wordline and bitline of a failing and a good 0.2 μm technology DRAM single cell were contacted. Both cells shared the same wordline for direct comparison of cell characteristics. Through this technique we electrically isolated the fail to a single cell by detecting leakage between the polysilicon wordline gate and the cell diffusion. For physical localization, we present a completely in-situ FIBSEM technique that combines ion milling, XeF2 staining and SEM imaging. With this technique, the electrically isolated fail was found to be a hole in the gate oxide at the bad cell.

Fabrication of Sub-250nm High Aspect Ratio Apertures by Focused Ion Beam Lithography

2006 ◽  
Vol 983 ◽  
Author(s):  
Todd Simpson ◽  
Ian V Mitchell
Keyword(s):  
Silicon Nitride ◽  
High Resolution ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Gold Films ◽  
Ion Beam Lithography ◽  
Diameter Hole ◽  
Sem Imaging ◽  
Aperture Arrays

AbstractAperture arrays were fabricated in 1.0µm thick gold films supported on 20nm thick silicon nitride membranes. Lithographic milling strategies in gold were evaluated through the use of in-situ sectioning and high resolution SEM imaging with the UWO CrossBeam FIB/SEM. A successful strategy for producing a 250nm diameter hole with sidewalls approaching vertical is summarized.

Applications of In-situ Sample Preparation and Modeling of SEM-STEM Imaging

2008 ◽  
Author(s):  
R.J. Young ◽  
A. Buxbaum ◽  
B. Peterson ◽  
R. Schampers
Keyword(s):  
Sample Preparation ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Dual Beam ◽  
Transmission Electron ◽  
Scanning Transmission ◽  
Electron Microscopes ◽  
Scanning Electron Microscopes ◽  
Preparation Techniques

Abstract Scanning transmission electron microscopy with scanning electron microscopes (SEM-STEM) has become increasing used in both SEM and dual-beam focused ion beam (FIB)-SEM systems. This paper describes modeling undertaken to simulate the contrast seen in such images. Such modeling provides the ability to help understand and optimize imaging conditions and also support improved sample preparation techniques.

A Preparation Method of Diamond Specimens Using an Advanced FIB Microscopy for Micro and Nanoanalysis

2012 ◽  
Vol 531-532 ◽  
pp. 592-595
Author(s):  
Yi Qing Chen ◽  
Feng Zai Tang ◽  
Liang Chi Zhang
Keyword(s):  
Focused Ion Beam ◽  
Preparation Method ◽  
Ion Beam ◽  
Polycrystalline Diamond ◽  
Specimen Preparation ◽  
Atomic Lattice ◽  
Cross Sectional ◽  
Site Specific ◽  
Dual Beam

This paper reports the specimen preparation using an advanced dual beam focused ion beam (FIB) technique for bulk polycrystalline diamond (PCD) composites after dynamic friction polishing (DFP). The technique adapted allows for precisely processing diamond materials at the specific polishing track sites of PCD surface, from which large cross-sectional specimens for SEM/EDS/Raman microanalysis could be successfully created. In addition, an in-situ lift-out method was developed to prepare the site-specific HRTEM specimens which were thin enough for imaging the atomic lattice of diamond and for conducting EELS analysis.

scholarly journals A streamlined workflow for automated cryo focused ion beam milling

2020 ◽  
Author(s):  
Sebastian Tacke ◽  
Philipp Erdmann ◽  
Zhexin Wang ◽  
Sven Klumpe ◽  
Michael Grange ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
High Vacuum ◽  
Software Application ◽  
Amorphous Ice ◽  
Cryo Electron Tomography ◽  
Structure Of Proteins ◽  
Cryogenic Conditions

AbstractCryo-electron tomography is an emerging technique to study the cellular architecture and the structure of proteins at high resolution in situ. Most biological specimens are too thick to be directly investigated and are therefore thinned by milling with a focused ion beam under cryogenic conditions. This procedure is prone to frost and amorphous ice depositions which makes it a tedious process, leading to suboptimal results especially when larger batches are milled. Here, we present new hardware that overcomes the current limitations. We developed a new glove box and a high vacuum cryo transfer system and installed a stage heater, a cryo-shield and a cryo-shutter in the FIB milling microscope. This tremendously reduces the ice depositions during transfer and milling, and simplifies the handling of the sample. In addition, we tested a new software application that automates the key milling steps. Together, these improvements allow for high-quality, high-throughput cryo-FIB milling.

Locally Condensed Water as a Solution for In Situ Wet Corrosion Electron Microscopy

2020 ◽  
Vol 26 (2) ◽  
pp. 211-219
Author(s):  
Majid Ahmadi ◽  
Frans D. Tichelaar ◽  
Andreas Ihring ◽  
Michael Kunze ◽  
Sophie Billat ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Water Layer ◽  
Ex Situ ◽  
Peltier Element ◽  
Tem Analysis ◽  
Dual Beam ◽  
Corrosion Studies

AbstractIn microstructural corrosion studies, knowledge on the initiation of corrosion on an nm-scale is lacking. In situ transmission electron microscope (TEM) studies can elucidate where/how the corrosion starts, provided that the proper corrosive conditions are present during the investigation. In wet corrosion studies with liquid cell nanoreactors (NRs), the liquid along the electron beam direction leads to strong scattering and therefore image blurring. Thus, a quick liquid removal or thickness control of the liquid layer is preferred. This can be done by the use of a Peltier element embedded in an NR. As a prelude to such in situ work, we demonstrate the local wetting of a TEM sample, by creating a temperature decrease of 10 ± 2°C on the membrane of an NR with planar Sb/BiSb thermoelectric materials for the Peltier element. TEM samples were prepared and loaded in an NR using a dual-beam focused ion beam scanning electron microscope. A mixture of water vapor and carrier gas was passed through a chamber, which holds the micro-electromechanical system Peltier device and resulted in quick formation of a water layer/droplets on the sample. The TEM analysis after repeated corrosion of the same sample (ex situ studies) shows the onset and progression of O2 and H2S corrosion of the AA2024-T3 alloy and cold-rolled HCT980X steel lamellae.

In-Situ Sample Preparation and High-Resolution SEM-STEM Analysis

2004 ◽  
Author(s):  
Richard J. Young ◽  
Michael P. Bernas ◽  
Mary V. Moore ◽  
Young-Chung Wang ◽  
Jay P. Jordan ◽  
...  
Keyword(s):  
Electron Microscope ◽  
High Resolution ◽  
Sample Preparation ◽  
Scanning Transmission Electron Microscopy ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Dual Beam ◽  
Transmission Electron ◽  
Scanning Transmission

Abstract The dual-beam system, which combines a high-resolution scanning electron microscope (SEM) with a focused ion beam (FIB), allows sample preparation, imaging, and analysis to be accomplished in a single tool. This paper discusses how scanning transmission electron microscopy (STEM) with the electron beam enhances the analysis capabilities of the dualbeam. In particular, it shows how, using the combination of in-situ sample preparation and integrated SEM-STEM imaging, more failure analysis and characterization problems can be solved in the dual-beam without needing to use the Ångstrom-level capabilities of the transmission electron microscope (TEM).

scholarly journals In situ Microfluidic Cryofixation for Cryo Focused Ion Beam Milling and Cryo Electron Tomography

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Marie Fuest ◽  
Miroslava Schaffer ◽  
Giovanni Marco Nocera ◽  
Rodrigo I. Galilea-Kleinsteuber ◽  
Jan-Erik Messling ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Light Microscope ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Light And Electron Microscopy ◽  
Live Imaging ◽  
Cryo Electron Tomography

AbstractWe present a microfluidic platform for studying structure-function relationships at the cellular level by connecting video rate live cell imaging with in situ microfluidic cryofixation and cryo-electron tomography of near natively preserved, unstained specimens. Correlative light and electron microscopy (CLEM) has been limited by the time required to transfer live cells from the light microscope to dedicated cryofixation instruments, such as a plunge freezer or high-pressure freezer. We recently demonstrated a microfluidic based approach that enables sample cryofixation directly in the light microscope with millisecond time resolution, a speed improvement of up to three orders of magnitude. Here we show that this cryofixation method can be combined with cryo-electron tomography (cryo-ET) by using Focused Ion Beam milling at cryogenic temperatures (cryo-FIB) to prepare frozen hydrated electron transparent sections. To make cryo-FIB sectioning of rapidly frozen microfluidic channels achievable, we developed a sacrificial layer technique to fabricate microfluidic devices with a PDMS bottom wall <5 µm thick. We demonstrate the complete workflow by rapidly cryo-freezing Caenorhabditis elegans roundworms L1 larvae during live imaging in the light microscope, followed by cryo-FIB milling and lift out to produce thin, electron transparent sections for cryo-ET imaging. Cryo-ET analysis of initial results show that the structural preservation of the cryofixed C. elegans was suitable for high resolution cryo-ET work. The combination of cryofixation during live imaging enabled by microfluidic cryofixation with the molecular resolution capabilities of cryo-ET offers an exciting avenue to further advance space-time correlative light and electron microscopy (st-CLEM) for investigation of biological processes at high resolution in four dimensions.

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