DOI: 10.1093/micmic/ozac029. Cryo-EXLO Manipulation of FIB Specimens for Cryo-TEM

Cryo-EXLO Manipulation of FIB Specimens for Cryo-TEM

10.1093/micmic/ozac029

Crossref journal-article

Oxford University Press (OUP)

Microscopy and Microanalysis (286)

Abstract

Abstract This work describes cryogenic ex situ lift out (cryo-EXLO) of cryogenic focused ion beam (cryo-FIB) thinned specimens for analysis by cryogenic transmission electron microscopy (cryo-TEM). The steps and apparatus necessary for cryo-EXLO are described. Methods designed to limit ice contamination include use of an anti-frost lid, a vacuum transfer assembly, and a cryostat. Cryo-EXLO is performed in a cryostat with the cryo-shuttle holder positioned in the cryogenic vapor phase above the surface of liquid N2 (LN2) using an EXLO manipulation station installed inside a glove box maintained at &lt; 10% relative humidity and inert (e.g., N2 gas) conditions. Thermal modeling shows that a cryo-EXLO specimen will remain vitreous within its FIB trench indefinitely while LN2 is continuously supplied. Once the LN2 is cut off, modeling shows that the EXLO specimen will remain vitreous for over 4 min, allowing sufficient time for the cryo-transfer steps which take only seconds to perform. Cryo-EXLO was applied successfully to cryo-FIB-milled specimen preparation of a polymer sample and plunge-frozen yeast cells. Cryo-TEM of both the polymer and the yeast shows minimal ice contamination with the yeast specimen maintaining its vitreous phase, illustrating the potential of cryo-EXLO for cryo-FIB-TEM of beam-sensitive, liquid, or biological materials.

Bibliography

Giannuzzi, L. A., Colletta, M., Yu, Y., Kourkoutis, L. F., Iams, A. D., Beggs, K., & Kassab, A. J. (2023). Cryo-EXLO Manipulation of FIB Specimens for Cryo-TEM. Microscopy and Microanalysis, 29(1), 145â154.

Authors 7

Lucille A Giannuzzi (first)
Michael Colletta (additional)
Yue Yu (additional)
Lena F Kourkoutis (additional)
Andrew D Iams (additional)
Kyle Beggs (additional)
Alain J Kassab (additional)

References 30 Referenced 10

{'key': '2023020121555061100_ozac029-B1', 'first-page': '399', 'article-title': 'Failure analysis of electronic material using cryogenic FIB-SEM', 'author': 'Antoniou', 'year': '2012'} / Failure analysis of electronic material using cryogenic FIB-SEM by Antoniou (2012)
10.1038/s41467-019-11015-0 / Nat Commun / Stabilizing polymer electrolytes in high-voltage lithium batteries by Choudhury (2019)
10.1111/j.1365-2818.1981.tb02483.x / J. Microsc / Vitrification of pure water for electron microscopy by Doubochet (1981)
10.1002/sia.1448 / Surf Interface Anal / XPS analysis of Si samples prepared by the FIB lift-out technique by Ferryman (2002)
10.1017/S1431927617009023 / Microsc Microanal / Simultaneous structural and electrical analysis of vanadium dioxide using in situ TEM by Ghassemi (2017)
{'key': '2023020121555061100_ozac029-B6', 'first-page': '275', 'article-title': 'Optimizing ex situ Lift Out for Frontside or Backside FIB Milling', 'author': 'Giannuzzi', 'year': '2017'} / Optimizing ex situ Lift Out for Frontside or Backside FIB Milling by Giannuzzi (2017)
10.1017/S1431927619008651 / Microsc Microanal / Micromanipulation, FIB, STEM, EDS and EELS of UF 4 particles by Giannuzzi (2019)
{'key': '2023020121555061100_ozac029-B8', 'first-page': '19', 'volume-title': 'Workshop on Specimen Preparation for TEM of Materials IV. Material Research Society Symposium Proceedings', 'author': 'Giannuzzi', 'year': '1997'} / Workshop on Specimen Preparation for TEM of Materials IV. Material Research Society Symposium Proceedings by Giannuzzi (1997)
10.1017/S1431927616001525 / Microsc Microanal / Manipulation of ceramic fibers to EXpressLO™ grids for FIB/TEM analysis by Giannuzzi (2016)
10.1007/0-387-23313-X_10 / Introduction to Focused Ion Beams: Instrumentation, Theory, Techniques, and Practice by Giannuzzi (2005)
10.1016/S0968-4328(99)00005-0 / Micron / A review of focused ion beam milling for TEM specimen preparation by Giannuzzi (1999)
10.1017/S1431927615013720 / Microsc Microanal / Theory and new applications of ex situ lift out by Giannuzzi (2015)
{'key': '2023020121555061100_ozac029-B9', 'first-page': '236', 'article-title': '3D-printed samples for EXLO/INLO practice and training', 'author': 'Giannuzzi', 'year': '2019'} / 3D-printed samples for EXLO/INLO practice and training by Giannuzzi (2019)
10.1016/S0091-679X(01)67003-1 / Methods Cell Biol / Using rapid freeze and freeze-substitution for the preparation of yeast cells for electron microscopy and three-dimensional analysis by Giddings (2001)
10.1111/j.1365-2818.2007.01775.x / J Microsc / A technique for improved focused ion beam milling of cryo-prepared life science specimens by Hayles (2007)
10.1016/j.jsb.2006.03.006 / J Struct Biol / Site-specific 3D imaging of cells and tissues with a dual beam microscope by Heymann (2006)
10.1016/j.jsb.2013.10.019 / J Struct Biol / Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography by Hsieh (2014)
10.7554/eLife.70506 / Elife / A modular platform for automated cryo-FIB workflows by Klumpe (2021)
{'key': '2023020121555061100_ozac029-B19', 'first-page': '211', 'volume-title': 'Cryo-EM Part A: Sample Preparation and Data Collection. Methods in Enzymology', 'author': 'Ladinsky', 'year': '2010'} / Cryo-EM Part A: Sample Preparation and Data Collection. Methods in Enzymology by Ladinsky (2010)
10.1016/j.chempr.2018.09.007 / Chem / Catalyst: how cryo-EM shapes the development of next-generation batteries by Li (2018)
10.1111/j.1365-2818.2006.01567.x / J. Microsc / Focused ion beam milling of vitreous water: Prospects for an alternative to cryo-ultramicrotomy of frozen-hydrated biological samples by Marko (2006)
10.1002/jemt.22630 / Microsc Res Tech / Making the practically impossible “merely difficult”—cryogenic FIB lift-out for “damage free” soft matter imaging by Parmenter (2016)
10.1111/jmi.12953 / J Microsc / Cryo-FIB-lift-out: practically impossible to practical reality by Parmenter (2021)
10.1073/pnas.1201333109 / Proc Nat Acad Sci USA / Focused ion beam micromachining of eukaryotic cells for cryoelectron tomography by Rigort (2012)
10.1016/j.jsb.2012.08.012 / J Struct Biol / A site-specific focused-ion-beam lift-out method for cryo transmission electron microscopy by Rubino (2012)
10.1017/S1431927618004592 / Microsc Microanal / Cryo-FIB lamella milling: A comprehensive technique to prepare samples of both plunge- and high-pressure frozen-hydrated specimens for in situ studies by Schaffer (2018)
10.1038/s41592-019-0497-5 / Nat Methods / A cryo-FIB lift-out technique enables molecular-resolution cryo-ET within native Caenorhabditis elegans tissue by Schaffer (2019)
10.1002/sia.1063 / Surf Interface Anal / Application of focused ion beam lift-out specimen preparation to TEM, SEM, STEM. AES and SIMS analysis by Stevie (2001)
10.1017/S1431927617012223 / Micros Microanal / Cryo-FIB milling and lift-out for preparation of specimens for cryo-TEM by Zachman (2017)
10.1038/s41586-018-0397-3 / Nature / Cryo-STEM mapping of solid–liquid interfaces and dendrites in lithium-metal batteries by Zachman (2018)

Dates

Type	When
Created	2 years, 7 months ago (Jan. 9, 2023, 2:47 a.m.)
Deposited	2 years, 6 months ago (Feb. 1, 2023, 10:26 p.m.)
Indexed	3 weeks, 6 days ago (July 24, 2025, 8:10 a.m.)
Issued	2 years, 7 months ago (Jan. 9, 2023)
Published	2 years, 7 months ago (Jan. 9, 2023)
Published Online	2 years, 7 months ago (Jan. 9, 2023)
Published Print	2 years, 6 months ago (Feb. 1, 2023)

Funders 3

DOE
Awards1
1. DE-SC0020511
NSF
Awards1
1. DMR-1654596, DMR-1429155
Cornell Center for Materials Research 10.13039/100008585
Region: Americas
pri (Universities (academic only))
Labels1
1. CCMR
Awards1
1. DMR-1719875

BibTeX

@article{Giannuzzi_2023, title={Cryo-EXLO Manipulation of FIB Specimens for Cryo-TEM}, volume={29}, ISSN={1435-8115}, url={http://dx.doi.org/10.1093/micmic/ozac029}, DOI={10.1093/micmic/ozac029}, number={1}, journal={Microscopy and Microanalysis}, publisher={Oxford University Press (OUP)}, author={Giannuzzi, Lucille A and Colletta, Michael and Yu, Yue and Kourkoutis, Lena F and Iams, Andrew D and Beggs, Kyle and Kassab, Alain J}, year={2023}, month=jan, pages={145–154} }

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