title: Localization of fluorescently labeled structures in frozen-hydrated samples using integrated light electron microscopy.
abstract: Correlative light and electron microscopy is an increasingly popular technique to study complex biological systems at various levels of resolution. Fluorescence microscopy can be employed to scan large areas to localize regions of interest which are then analyzed by electron microscopy to obtain morphological and structural information from a selected field of view at nm-scale resolution. Previously, an integrated approach to room temperature correlative microscopy was described. Combined use of light and electron microscopy within one instrument greatly simplifies sample handling, avoids cumbersome experimental overheads, simplifies navigation between the two modalities, and improves the success rate of image correlation. Here, an integrated approach for correlative microscopy under cryogenic conditions is presented. Its advantages over the room temperature approach include safeguarding the native hydrated state of the biological specimen, preservation of the fluorescence signal without risk of quenching due to heavy atom stains, and reduced photo bleaching. The potential of cryo integrated light and electron microscopy is demonstrated for the detection of viable bacteria, the study of in vitro polymerized microtubules, the localization of mitochondria in mouse embryonic fibroblasts, and for a search into virus-induced intracellular membrane modifications within mammalian cells.
authors.affiliation: Department of Molecular Cell Biology, Section Electron Microscopy, Leiden University Medical Center, P.O. Box 9600, 2300 RC, Leiden, The Netherlands.
authors.forename: F G A
authors.initials: FG
authors.lastname: Faas
authors.forename: M
authors.initials: M
authors.lastname: Bárcena
authors.forename: A V
authors.initials: AV
authors.lastname: Agronskaia
authors.forename: H C
authors.initials: HC
authors.lastname: Gerritsen
authors.forename: K B
authors.initials: KB
authors.lastname: Moscicka
authors.forename: C A
authors.initials: CA
authors.lastname: Diebolder
authors.forename: L F
authors.initials: LF
authors.lastname: van Driel
authors.forename: R W A L
authors.initials: RW
authors.lastname: Limpens
authors.forename: E
authors.initials: E
authors.lastname: Bos
authors.forename: R B G
authors.initials: RB
authors.lastname: Ravelli
authors.forename: R I
authors.initials: RI
authors.lastname: Koning
authors.forename: A J
authors.initials: AJ
authors.lastname: Koster
mesh_terms: D040981:Acinetobacter baumannii; D000818:Animals; D002522:Chlorocebus aethiops; D020285:Cryoelectron Microscopy / Q000379:methods*; D005453:Fluorescence; D005615:Freezing; D008854:Microscopy, Electron / Q000379:methods*; D008856:Microscopy, Fluorescence; D008870:Microtubules; D013048:Specimen Handling / Q000379:methods*; D014709:Vero Cells