ausblenden:
Schlagwörter:
Cryo-fluorescence microscopy (cryo-FM)
Correlative light and electron
microscopy(CLEM)
Cryo-electron microscopy (cryo-EM)
High accuracy
localization
Fiducial beads
Bacteriophage particles
Low-temperature
fluorescence microscopy
light-electron-microscopy
plasma-membrane
tomography
protein
ultrastructure
specimen
embryos
cells
Microscopy
Zusammenfassung:
Performing fluorescence microscopy and electron microscopy on the same sample allows fluorescent signals to be used to identify and locate features of interest for subsequent imaging by electron microscopy. To carry out such correlative microscopy on vitrified samples appropriate for structural cryoelectron microscopy it is necessary to perform fluorescence microscopy at liquid-nitrogen temperatures. Here we describe an adaptation of a cryo-light microscopy stage to permit use of high-numerical aperture objectives. This allows high-sensitivity and high-resolution fluorescence microscopy of vitrified samples. We describe and apply a correlative cryo-fluorescence and cryo-electron microscopy workflow together with a fiducial bead-based image correlation procedure. This procedure allows us to locate fluorescent bacteriophages in cryo-electron microscopy images with an accuracy on the order of 50 nm, based on their fluorescent signal. It will allow the user to precisely and unambiguously identify and locate objects and events for subsequent high-resolution structural study, based on fluorescent signals. (C) 2013 Elsevier B.V. All rights reserved.
