Cryo-Focused Ion Beam Lamella Preparation Protocol for in Situ Structural Biology

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.

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Protocol for cryo-focused ion beam lift-out technique

10.21203/rs.2.10392/v1 ◽

2019 ◽

Cited By ~ 1

Author(s):

Miroslava Schaffer ◽

Stefan Pfeffer ◽

Julia Mahamid ◽

Stephan Kleindiek ◽

Tim Laugks ◽

...

Keyword(s):

High Pressure ◽

Structural Biology ◽

Focused Ion Beam ◽

Preparation Method ◽

Electron Tomography ◽

Ion Beam ◽

Cell Interior ◽

Cryo Electron Tomography ◽

Multicellular Organisms

Abstract Cryo-focused ion beam milling of frozen hydrated cells for the production of thin lamellas in combination with cryo-electron tomography (cryo-ET) has yielded unprecedented insights into the cell interior. This method allows access to native structures deep inside cells, enabling structural studies of macromolecules in situ. However, it is only suitable for cells that can be vitrified by plunge freezing (<10 μm). Multicellular organisms and tissues are considerably thicker and high-pressure freezing is required to ensure optimal preservation. Here, we describe a preparation method for extracting lamellas from high pressure frozen samples with a new cryo-gripper tool. This in situ lift-out technique at cryo-temperatures enables cryo-ET to be performed on multicellular organisms and tissue, extending the range of applications for in situ structural biology.

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

ISTFA 2014: Conference Proceedings from the 40th International Symposium for Testing and Failure Analysis ◽

10.31399/asm.cp.istfa2014p0400 ◽

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.

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Investigations of Leakage Paths in Sub-0.35μm DRAM Products Using Advanced Focused Ion Beam Techniques

ISTFA 1998: Conference Proceedings from the 24th International Symposium for Testing and Failure Analysis ◽

10.31399/asm.cp.istfa1998p0289 ◽

1998 ◽

Author(s):

H. Lorenz ◽

C. Engel

Keyword(s):

Focused Ion Beam ◽

Cell Function ◽

Dielectric Breakdown ◽

Ion Beam ◽

Electrical Characterization ◽

Floating Gate ◽

Leakage Path ◽

Contrast Technique ◽

Voltage Contrast

Abstract Due to the continuously decreasing cell size of DRAMs and concomitantly diminishing thickness of some insulating layers new failure mechanisms appear which until now had no significance for the cell function. For example high resistance leakage paths between closely spaced conductors can lead to retention problems. These are hard to detect by electrical characterization in a memory tester because the involved currents are in the range of pA. To analyze these failures we exploit the very sensitive passive voltage contrast of the Focused Ion Beam Microscope (FIB). The voltage contrast can further be enhanced by in-situ FIB preparations to obtain detailed information about the failure mechanism. The first part of this paper describes a method to detect a leakage path between a borderless contact on n-diffusion and an adjacent floating gate by passive voltage contrast achieved after FIB circuit modification. In the second part we will demonstrate the localization of a DRAM trench dielectric breakdown. In this case the FIB passive voltage contrast technique is not limited to the localization of the failing trench. We can also obtain the depth of the leakage path by selective insitu etching with XeF2 stopped immediately after a voltage contrast change.

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Cyclic Deformation of Microcantilevers Using In-Situ Micromanipulation

Experimental Mechanics ◽

10.1007/s11340-021-00752-3 ◽

2021 ◽

Author(s):

A. H. S. Iyer ◽

M. H. Colliander

Keyword(s):

Degrees Of Freedom ◽

Focused Ion Beam ◽

Ion Beam ◽

Structural Components ◽

Cyclic Testing ◽

Phase Boundaries ◽

Bulk Specimen ◽

Arbitrary Position ◽

Three Degrees Of Freedom

Abstract Background The trend in miniaturisation of structural components and continuous development of more advanced crystal plasticity models point towards the need for understanding cyclic properties of engineering materials at the microscale. Though the technology of focused ion beam milling enables the preparation of micron-sized samples for mechanical testing using nanoindenters, much of the focus has been on monotonic testing since the limited 1D motion of nanoindenters imposes restrictions on both sample preparation and cyclic testing. Objective/Methods In this work, we present an approach for cyclic microcantilever bending using a micromanipulator setup having three degrees of freedom, thereby offering more flexibility. Results The method has been demonstrated and validated by cyclic bending of Alloy 718plus microcantilevers prepared on a bulk specimen. The experiments reveal that this method is reliable and produces results that are comparable to a nanoindenter setup. Conclusions Due to the flexibility of the method, it offers straightforward testing of cantilevers manufactured at arbitrary position on bulk samples with fully reversed plastic deformation. Specific microstructural features, e.g., selected orientations, grain boundaries, phase boundaries etc., can therefore be easily targeted.

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Cryo-focused-ion-beam applications in structural biology

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2015.02.009 ◽

2015 ◽

Vol 581 ◽

pp. 122-130 ◽

Cited By ~ 66

Author(s):

Alexander Rigort ◽

Jürgen M. Plitzko

Keyword(s):

Structural Biology ◽

Focused Ion Beam ◽

Ion Beam

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

MRS Proceedings ◽

10.1557/proc-983-0983-ll05-06 ◽

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.

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Thermal stability of Pt nanowires manufactured by Ga+ focused ion beam (FIB)

MRS Proceedings ◽

10.1557/proc-777-t2.3 ◽

2003 ◽

Vol 777 ◽

Cited By ~ 2

Author(s):

B.J. Inkson ◽

G. Dehm

Keyword(s):

Thermal Stability ◽

Focused Ion Beam ◽

Ion Beam ◽

Wire Surface ◽

Cross Sectional ◽

Partial Crystallization ◽

Fcc Structure ◽

Thermal Stability Of ◽

Pt Nanowires

AbstractPt nanowires have been produced by FIB deposition of Pt thin films in a commercial Ga+ focused ion beam (FIB) system, followed by cross-sectional sputtering to form electron transparent Pt nanowires. The thermal stability of amorphous FIB manufactured Pt wires has been investigated by in-situ thermal cycling in a TEM. The Pt wires are stable up to 580-650°C where partial crystallization is observed in vacuum. Facetted nanoparticles grow on the wire surface, growing into free space by surface diffusion and minimising contact area with the underlying wire. The particles are fcc Pt with some dissolved Ga. Continued heating results in particle spheroidization, coalescence and growth, retaining the fcc structure.

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Fiducial marker application method for position alignment of in situ multimodal X-ray experiments and reconstructions

Journal of Applied Crystallography ◽

10.1107/s1600576716001989 ◽

2016 ◽

Vol 49 (2) ◽

pp. 700-704 ◽

Cited By ~ 12

Author(s):

Paul A. Shade ◽

David B. Menasche ◽

Joel V. Bernier ◽

Peter Kenesei ◽

Jun-Sang Park ◽

...

Keyword(s):

Focused Ion Beam ◽

Ion Beam ◽

Tensile Specimen ◽

Material Behavior ◽

Validation Data ◽

Great Opportunity ◽

Characterization Methods ◽

X Ray ◽

Multiple Data Sets

An evolving suite of X-ray characterization methods are presently available to the materials community, providing a great opportunity to gain new insight into material behavior and provide critical validation data for materials models. Two critical and related issues are sample repositioning during an in situ experiment and registration of multiple data sets after the experiment. To address these issues, a method is described which utilizes a focused ion-beam scanning electron microscope equipped with a micromanipulator to apply gold fiducial markers to samples for X-ray measurements. The method is demonstrated with a synchrotron X-ray experiment involving in situ loading of a titanium alloy tensile specimen.

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