Abstract
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.
Highlights
IntroductionElectron cryo microscopy (cryoEM) is a powerful technique to visualise molecules in their native state
Electron cryo microscopy is a powerful technique to visualise molecules in their native state
Adenovirus particles were labelled with Alexa-488 fluorophores (Ad5-488, for details see Section 2) providing a biological probe of known structure with a photon emission signal in the green spectrum [36,37]
Summary
Electron cryo microscopy (cryoEM) is a powerful technique to visualise molecules in their native state. Vitrification, i.e. rapid freezing of specimens within an amorphous, non-crystalline, glass-like ice layer, allows the preservation of structures down to the molecular level in the native, hydrated state. This is of special importance for cellular samples that are sensitive to chemical fixation and dehydration [4,5]. CryoEM has been used to study a vast range of specimens, from isolated macromolecules to the complexity of a cell Despite these tremendous capabilities, particular for cellular cryoEM, one major limitation remains: locating the subcellular event of interest with nanometre-scale precision on the three-millimetre-diameter, frozen-hydrated, EM grid
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