Cryo-FIB preparation of whole cells and tissue for cryo-TEM: use of high-pressure frozen specimens in tubes and planchets.

Journal of Microscopy
D A Matthijs de WinterMichael F Hayles

Abstract

The desire to study macromolecular complexes within their cellular context requires the ability to produce thin samples suitable for cryo-TEM (cryo-transmission electron microscope) investigations. In this paper, we discuss two similar approaches, which were developed independently in Utrecht (the Netherlands) and Albany (USA). The methods are particularly suitable for both tissue samples and cell suspensions prepared by a high-pressure freezer (HPF). The workflows are explained with particular attention to potential pitfalls, while underlying principles are highlighted ('why to do so'). Although both workflows function with a high success rate, full execution requires considerable experience and remains demanding. In addition, throughput is low. We hope to encourage other research groups worldwide to take on the challenge of improving the HPF- cryo-FIB-SEM - cryo-TEM workflow. We discuss a number of suggestions to this end. LAY DESCRIPTION: Life is ultimately dictated by the interaction of molecules in our bodies. Highly complex equipment is being used and further developed to study these interactions. The present paper describes methods to prepare small, very thin lamellae (area of 5×5 µm2 , thickness 50-300 nm) of a cell to ...Continue Reading

References

Nov 1, 1992·Journal of Microscopy·E KellenbergerW Villiger
May 1, 1988·Quarterly Reviews of Biophysics·J DubochetP Schultz
Oct 1, 1982·Cell Biology International Reports·C H Wouters, H K Koerten
Sep 1, 1995·Journal of Microscopy·D StuderM D Buschmann
Mar 1, 2007·Methods in Cell Biology·Kent McDonald
Jul 31, 2008·Journal of Structural Biology·Alexandra V AgronskaiaHans C Gerritsen
Jan 19, 2010·Proceedings of the National Academy of Sciences of the United States of America·Alke MeentsClemens Schulze-Briese
Feb 25, 2010·Journal of Structural Biology·Alexander RigortJürgen M Plitzko
Jul 20, 2010·Journal of Structural Biology·Michael F HaylesArie J Verkleij
Sep 28, 2010·Methods in Cell Biology·Wiebke MöbiusFrédérique Varoqueaux
Nov 30, 2010·Micron : the International Research and Review Journal for Microscopy·M A AronovaR D Leapman
Dec 16, 2010·Journal of Microscopy·I WeikusatM R Drury
Apr 29, 2011·Journal of Synchrotron Radiation·Manikandan KaruppasamyRaimond B G Ravelli
Mar 7, 2012·Proceedings of the National Academy of Sciences of the United States of America·Alexander RigortJürgen M Plitzko
Sep 25, 2012·Journal of Structural Biology·Stefano RubinoKlaus Leifer
Jul 31, 2013·Journal of Microscopy·A C ZonnevylleJ P Hoogenboom
Oct 15, 2013·Journal of Structural Biology·Andreas SchertelWiebke Möbius
Nov 12, 2013·Journal of Structural Biology·Hirofumi IijimaKuniaki Nagayama
Nov 12, 2013·Journal of Structural Biology·Chyongere HsiehMichael Marko
Sep 1, 2014·Matrix Biology : Journal of the International Society for Matrix Biology·Ernst B HunzikerNahoko Shintani
Jun 9, 2015·European Journal of Translational Myology·Terence WagenknechtMichael Marko
Oct 13, 2015·Journal of Molecular Biology·Shoh AsanoWolfgang Baumeister
Jan 16, 2016·Biophysical Journal·Jan ArnoldJürgen M Plitzko
Feb 16, 2016·Microscopy Research and Technique·Christopher D J ParmenterHoda M Eltaher
Feb 16, 2016·Journal of Structural Biology·Jianguo ZhangFei Sun
Feb 26, 2016·European Journal of Translational Myology·Terence WagenknechtMichael Marko
Apr 26, 2016·Nature Methods·Allison Doerr
Jul 23, 2016·Journal of Structural Biology·Miroslava SchafferJürgen M Plitzko
Jul 21, 2017·FEBS Letters·Jonathan WagnerRubén Fernández-Busnadiego
Jul 2, 2019·ELife·Sergey GorelickAlex de Marco

❮ Previous
Next ❯

Citations

Aug 2, 2020·Journal of Microscopy·M F Hayles, D A M DE Winter
Aug 21, 2020·Journal of Microscopy·Christopher David Parmenter, Zubair Ahmed Nizamudeen
Nov 1, 2020·International Journal of Molecular Sciences·Yiu Wing Sunny CheungCalvin K Yip

❮ Previous
Next ❯

Methods Mentioned

BETA
electron microscopy
light microscopy
electron diffraction

Software Mentioned

Quorum

Related Concepts

Trending Feeds

COVID-19

Coronaviruses encompass a large family of viruses that cause the common cold as well as more serious diseases, such as the ongoing outbreak of coronavirus disease 2019 (COVID-19; formally known as 2019-nCoV). Coronaviruses can spread from animals to humans; symptoms include fever, cough, shortness of breath, and breathing difficulties; in more severe cases, infection can lead to death. This feed covers recent research on COVID-19.

Blastomycosis

Blastomycosis fungal infections spread through inhaling Blastomyces dermatitidis spores. Discover the latest research on blastomycosis fungal infections here.

Nuclear Pore Complex in ALS/FTD

Alterations in nucleocytoplasmic transport, controlled by the nuclear pore complex, may be involved in the pathomechanism underlying multiple neurodegenerative diseases including Amyotrophic Lateral Sclerosis and Frontotemporal Dementia. Here is the latest research on the nuclear pore complex in ALS and FTD.

Applications of Molecular Barcoding

The concept of molecular barcoding is that each original DNA or RNA molecule is attached to a unique sequence barcode. Sequence reads having different barcodes represent different original molecules, while sequence reads having the same barcode are results of PCR duplication from one original molecule. Discover the latest research on molecular barcoding here.

Chronic Fatigue Syndrome

Chronic fatigue syndrome is a disease characterized by unexplained disabling fatigue; the pathology of which is incompletely understood. Discover the latest research on chronic fatigue syndrome here.

Evolution of Pluripotency

Pluripotency refers to the ability of a cell to develop into three primary germ cell layers of the embryo. This feed focuses on the mechanisms that underlie the evolution of pluripotency. Here is the latest research.

Position Effect Variegation

Position Effect Variagation occurs when a gene is inactivated due to its positioning near heterochromatic regions within a chromosome. Discover the latest research on Position Effect Variagation here.

STING Receptor Agonists

Stimulator of IFN genes (STING) are a group of transmembrane proteins that are involved in the induction of type I interferon that is important in the innate immune response. The stimulation of STING has been an active area of research in the treatment of cancer and infectious diseases. Here is the latest research on STING receptor agonists.

Microbicide

Microbicides are products that can be applied to vaginal or rectal mucosal surfaces with the goal of preventing, or at least significantly reducing, the transmission of sexually transmitted infections. Here is the latest research on microbicides.

Related Papers

Microscopy and Microanalysis : the Official Journal of Microscopy Society of America, Microbeam Analysis Society, Microscopical Society of Canada
Nathan D Burrows, R Lee Penn
© 2021 Meta ULC. All rights reserved