title: The making of frozen-hydrated, vitreous lamellas from cells for cryo-electron microscopy.
abstract: There has been a long standing desire to produce thick (up to 500 nm) cryo-sections of fully hydrated cells and tissue for high-resolution analysis in their natural state by cryo-transmission electron microscopy. Here, we present a method that can successfully produce sections (lamellas in FIB-SEM terminology) of fully hydrated, unstained cells from high-pressure frozen samples by focused ion beam (FIB) milling. The samples are therefore placed in thin copper tubes and vitrified by high-pressure freezing. For transfer, handling and subsequent milling, the tubes are placed in a novel connective device (ferrule) that protects the sample from devitrification and contamination and passes through all operation steps. A piezo driven sample positioning stage (cryo-nano-bench, CNB) with three degrees of freedom was additionally developed to enable accurate milling of frozen-hydrated lamellas. With the CNB, high-pressure frozen samples can be milled to produce either thin lamellas (<100 nm), for direct imaging by high-resolution cryo-TEM or thicker lamellas (300-500 nm) for cryo-electron tomography. The sample remains vitreous throughout the process by using the presented tools and methods. The results are an important step towards investigating larger cells and even tissue in there natural state which in the end will enable us to gain better insights into cellular processes.
authors.affiliation: FEI Company, Achtseweg Noord 5, 5600 KA Eindhoven, The Netherlands. Mike.hayles@fei.com
authors.forename: Michael F
authors.initials: MF
authors.lastname: Hayles
authors.forename: D A Matthijs
authors.initials: DA
authors.lastname: de Winter
authors.forename: Chris T W M
authors.initials: CT
authors.lastname: Schneijdenberg
authors.forename: Johannes D
authors.initials: JD
authors.lastname: Meeldijk
authors.forename: Uwe
authors.initials: U
authors.lastname: Luecken
authors.forename: Hans
authors.initials: H
authors.lastname: Persoon
authors.forename: Jeroen
authors.initials: J
authors.lastname: de Water
authors.forename: Frank
authors.initials: F
authors.lastname: de Jong
authors.forename: Bruno M
authors.initials: BM
authors.lastname: Humbel
authors.forename: Arie J
authors.initials: AJ
authors.lastname: Verkleij
mesh_terms: D020285:Cryoelectron Microscopy / Q000295:instrumentation / Q000379:methods*; D055032:Electron Microscope Tomography / Q000379:methods; D005629:Frozen Sections / Q000295:instrumentation* / Q000379:methods*; D046529:Microscopy, Electron, Transmission / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000648:ultrastructure