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Correlative light and electron microscopy (CLEM) is an emerging technique which combines functional information provided by fluorescence microscopy (FM) with the high-resolution structural information of electron microscopy (EM). So far, correlative cryo microscopy of frozen-hydrated samples has not reached better than micrometre range accuracy. Here, a method is presented that enables the correlation between fluorescently tagged proteins and electron cryo tomography (cryoET) data with nanometre range precision. Specifically, thin areas of vitrified whole cells are examined by correlative fluorescence cryo microscopy (cryoFM) and cryoET. Novel aspects of the presented cryoCLEM workflow not only include the implementation of two independent electron dense fluorescent markers to improve the precision of the alignment, but also the ability of obtaining an estimate of the correlation accuracy for each individual object of interest. The correlative workflow from plunge-freezing to cryoET is detailed step-by-step for the example of locating fluorescence-labelled adenovirus particles trafficking inside a cell.

Original publication

DOI

10.1016/j.ultramic.2013.10.011

Type

Journal article

Journal

Ultramicroscopy

Publication Date

08/2014

Volume

143

Pages

41 - 51

Keywords

Cellular tomography, Correlative microscopy, Electron cryo microscopy, Fluorescent cryo microscopy, Fluorescent microspheres, Cell Line, Cryoelectron Microscopy, Electrons, Fluorescence, Fluorescent Dyes, HEK293 Cells, Humans, Microscopy, Fluorescence, Microscopy, Polarization