Protocol for cryo-focused ion beam lift-out technique

2019 ◽  
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.

scholarly journals VHUT-cryo-FIB, a method to fabricate frozen-hydrated lamella of tissue specimen for in situ cryo-electron tomography

2019 ◽  
Author(s):  
Jianguo Zhang ◽  
Danyang Zhang ◽  
Lei Sun ◽  
Gang Ji ◽  
Xiaojun Huang ◽  
...  
Keyword(s):  
High Pressure ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Tissue Sample ◽  
Ion Beam ◽  
Tissue Specimen ◽  
High Pressure Freezing ◽  
Tissue Samples ◽  
Cryo Electron Tomography

ABSTACTCryo-electron tomography (cryo-ET) provides a promising technique to study high resolution structures of macromolecules in situ, opening a new era of structural biology. One major bottleneck of this technique is to prepare suitable cryo-lamellas of cell/tissue samples. The emergence of cryo-focused ion beam (cryo-FIB) milling technique provides a good solution of this bottleneck. However, there are still large limitations of using cryo-FIB to prepare cryo-lamella of tissue specimen because the thickness of tissue increases the difficulty of specimen freezing and cryo-FIB milling. Here we report a new workflow, VHUT-cryo-FIB (Vibratome - High pressure freezing - Ultramicrotome Trimming – cryo-FIB), aiming for efficient preparation of frozen hydrated tissue lamella for subsequent cryo-ET data collection. This workflow includes tissue slicing using vibratome, high pressure freezing, ultramicrotome cryo-trimming, cryo-FIB milling and the subsequent cryo-electron microscopy (cryo-EM). The modification of equipment in this workflow is highly eliminated. We developed two strategies with a special cryo-holder tip or carrier for loading cryo-lamella into side entry cryo-holder or Autoloader catridge. We tested this workflow using the tissue sample of rat skeleton muscle and spinach leaf and collected high quality cryo-ET tilt series, which enabled us to obtain an in situ structure of spinach ribosome by sub-tomogram averaging.

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 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.

scholarly journals PIE-scope, integrated cryo-correlative light and FIB/SEM microscopy

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Sergey Gorelick ◽  
Genevieve Buckley ◽  
Gediminas Gervinskas ◽  
Travis K Johnson ◽  
Ava Handley ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Fluorescent Proteins ◽  
Electron Tomography ◽  
Protein Complexes ◽  
Ion Beam ◽  
Correlative Microscopy ◽  
New Approach ◽  
Macromolecular Complexes ◽  
Cryo Electron Tomography

Cryo-electron tomography (cryo-ET) is emerging as a revolutionary method for resolving the structure of macromolecular complexes in situ. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-focused ion beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. Specifically, we demonstrate the versatility of PIE-scope by preparing targeted cryo-lamellae from subcellular compartments of neurons from transgenic Caenorhabditis elegans and Drosophila melanogaster expressing fluorescent proteins. We designed PIE-scope to enable retrofitting of existing microscopes, which will increase the throughput and accuracy on projects requiring correlative microscopy to target protein complexes. This new approach will make cryo-correlative workflow safer and more accessible.

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.

scholarly journals Integrated Cryo-Correlative Microscopy for Targeted Structural Investigation In Situ

2021 ◽  
Vol 29 (6) ◽  
pp. 20-25
Author(s):  
Marit Smeets ◽  
Anna Bieber ◽  
Cristina Capitanio ◽  
Oda Schioetz ◽  
Thomas van der Heijden ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Building Blocks ◽  
Quality Data ◽  
Cellular Interaction ◽  
High Quality Data ◽  
Cryo Electron Tomography ◽  
Sample Transfer

Abstract:Cryo-electron tomography (cryo-ET) has the potential to revolutionize our understanding of the building blocks of life since it provides the unique opportunity to study molecules and membrane architectures in the context of cellular interaction. In particular, the combination of fluorescence imaging with focused ion beam (FIB) milling allows the targeting of specific structures in thick cellular samples by preparing thin lamellae that contain a specific fluorescence marker. This technique has conventionally been time-consuming, as it requires sample transfer to multiple microscopes and presents several technical challenges that currently limit its success. Here we describe METEOR, a FIB-integrated microscopy solution that streamlines the correlative cryo-ET workflow. It protects the sample from ice contamination by minimizing handling steps, thus increasing the likelihood of high-quality data. It also allows for monitoring of the milling procedure to ensure the molecule of interest is captured and can then be imaged by cryo-ET.

scholarly journals Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

2022 ◽  
Author(s):  
Jie E Yang ◽  
Matthew R Larson ◽  
Bryan S Sibert ◽  
Joseph Y Kim ◽  
Daniel Parrell ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Low Dose ◽  
Structural Information ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensional ◽  
Structural Studies ◽  
Cryo Electron Tomography ◽  
Efficient Data

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here, we present robust tools for montage electron tomography tailored for vitrified specimens. The integration of correlative cryo-fluorescence microscopy, focused-ion beam milling, and micropatterning produces contextual three-dimensional architecture of cells. Montage tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

scholarly journals Cryo-electron tomography of native Drosophila tissues vitrified by plunge freezing

2021 ◽  
Author(s):  
Felix J.B. Baeuerlein ◽  
Jose C. Pastor-Pareja ◽  
Ruben Fernandez-Busnadiego
Keyword(s):  
High Pressure ◽  
High Resolution ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
High Resolution Imaging ◽  
Molecular Architecture ◽  
High Pressure Freezing ◽  
Cryo Electron Tomography ◽  
Resolution Imaging

Cryo-focused ion beam (cryo-FIB) milling allows thinning vitrified cells for high resolution imaging by cryo-electron tomography (cryo-ET). However, it remains challenging to apply this workflow to tissues, as they usually require high-pressure freezing for vitrification. Here we show that dissected Drosophila tissues can be directly vitrified by plunge freezing upon a short incubation in 10% glycerol. This expedites subsequent cryo-FIB/ET, enabling systematic analyses of the molecular architecture of native tissues.

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.

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.

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