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Thinning of large mammalian cells for cryo-TEM characterization by cryo-FIB milling. 2012

K M Strunk, and K Wang, and D Ke, and J L Gray, and P Zhang
Department of Mechanical Engineering and Materials Science, Swanson School of Engineering, University of Pittsburgh, Pittsburgh, Pennsylvania 15261, USA.

Focused ion beam milling at cryogenic temperatures (cryo-FIB) is a valuable tool that can be used to thin vitreous biological specimens for subsequent imaging and analysis by cryo-transmission electron microscopy (cryo-TEM) in a frozen-hydrated state. This technique offers the potential benefit of eliminating the mechanical artefacts that are typically found with cryo-ultramicrotomy. However, due to the additional complexity in transferring samples in and out of the FIB, contamination and devitrification of the amorphous ice is commonly encountered. To address these problems, we have designed a sample cryo-shuttle that directly and specifically accepts Polara TEM cartridges to simplify the transfer process between FIB and TEM. We optimized several parameters in the cryo-FIB and cryo-TEM processes using the quality of the samples' ice as an indicator and demonstrated high-quality milling with large mammalian cells. By comparing the results from HeLa cells to those from Escherichia coli cells, we discuss some of the artefacts and challenges we have encountered using this technique.

UI MeSH Term Description Entries
D007089 Image Enhancement Improvement of the quality of a picture by various techniques, including computer processing, digital filtering, echocardiographic techniques, light and ultrastructural MICROSCOPY, fluorescence spectrometry and microscopy, scintigraphy, and in vitro image processing at the molecular level. Image Quality Enhancement,Enhancement, Image,Enhancement, Image Quality,Enhancements, Image,Enhancements, Image Quality,Image Enhancements,Image Quality Enhancements,Quality Enhancement, Image,Quality Enhancements, Image
D008322 Mammals Warm-blooded vertebrate animals belonging to the class Mammalia, including all that possess hair and suckle their young. Mammalia,Mammal
D004926 Escherichia coli A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc. Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D006367 HeLa Cells The first continuously cultured human malignant CELL LINE, derived from the cervical carcinoma of Henrietta Lacks. These cells are used for, among other things, VIRUS CULTIVATION and PRECLINICAL DRUG EVALUATION assays. Cell, HeLa,Cells, HeLa,HeLa Cell
D006801 Humans Members of the species Homo sapiens. Homo sapiens,Man (Taxonomy),Human,Man, Modern,Modern Man
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D013048 Specimen Handling Procedures for collecting, preserving, and transporting of specimens sufficiently stable to provide accurate and precise results suitable for clinical interpretation. Specimen Collection,Collection, Specimen,Collections, Specimen,Handling, Specimen,Handlings, Specimen,Specimen Collections,Specimen Handlings
D015925 Cryopreservation Preservation of cells, tissues, organs, or embryos by freezing. In histological preparations, cryopreservation or cryofixation is used to maintain the existing form, structure, and chemical composition of all the constituent elements of the specimens. Cryofixation,Cryonic Suspension,Cryonic Suspensions,Suspension, Cryonic
D016477 Artifacts Any visible result of a procedure which is caused by the procedure itself and not by the entity being analyzed. Common examples include histological structures introduced by tissue processing, radiographic images of structures that are not naturally present in living tissue, and products of chemical reactions that occur during analysis. Artefacts,Artefact,Artifact
D016616 Cryoultramicrotomy The technique of using a cryostat or freezing microtome, in which the temperature is regulated to -20 degrees Celsius, to cut ultrathin frozen sections for microscopic (usually, electron microscopic) examination. Cryosectioning,Freeze Sectioning,Sectioning, Freeze

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