Correlative light and electron microscopy methods for the study of virus–cell interactions

Abstract

Electron microscopy (EM) is an invaluable tool to study the interactions of viruses with cells, and the ultrastructural changes induced in host cells by virus infection. Light microscopy (LM) is a complementary tool with the potential to locate rare events, label specific components, and obtain dynamic information. The combination of LM and EM in correlative light and electron microscopy (CLEM) is particularly powerful. It can be used to complement a static EM image with dynamic data from live imaging, identify the ultrastructure observed in LM, or, conversely, provide molecular specificity data for a known ultrastructure. Here, we describe methods and strategies for CLEM, discuss their advantages and limitations, and review applications of CLEM to study virus-host interactions.Keywords: correlative light and electron microscopy; electron microscopy; virus-host interaction Viruses are obligate intracellular parasites whose replication is dependent on the intimate interaction with the host cell. Virus infection starts upon target cell engagement, generally through the recognition of attachment factors and specific receptor molecules on the cell surface. A combination of intrinsic features, such as virus size and the presence of a lipid envelope, with specific factors, such as the ability to recruit distinct host receptors, determine the type of internalization process. Whatever the entry route is, that is, endocytosis or direct fusion at the plasma membrane, the result is the release of the viral genome, often bound to viral proteins, into the cytoplasm. There, the viral genome must reach the appropriate intracellular Abbreviations CEMOVIS, cryo-electron microscopy of vitreous sections; CLEM, correlative light and electron

Distinguishing signal from autofluorescence in cryogenic correlated light and electron microscopy of mammalian cells

“…To facilitate image correlation, we added 500 nm blue fluorospheres to the samples before plunge-freezing [as in (Bykov et al, 2016, Schorb and Briggs, 2014, Chang et al, 2014, Liu et al, 2015, Schellenberger et al, 2014)]. Significantly, these fluorospheres were visible in both cryo-LM and cryo-EM modalities.…”

“…Cryo-CLEM experiments on mammalian cells have been previously performed using organic fluorescent dyes or proteins tagged with fluorophores (Kaufmann et al, 2014, Liu et al, 2015, Schorb and Briggs, 2014, Schorb et al, 2016, Bykov et al, 2016, Schellenberger et al, 2014, Jun et al, 2011). Though organic dyes are very bright, it can be challenging to use these compounds to efficiently label specific proteins inside cells.…”

Automated detection of fluorescent cells in in‐resin fluorescence sections for integrated light and electron microscopy

“…One of these methods is known as array tomography, whereby an array of ultrathin sections cut through resin‐embedded cells or tissues are imaged sequentially with a scanning electron microscope (SEM) to build up a 3D stack of images through the volume (Micheva & Smith, ; Wacker & Schroeder, ; Hayworth et al ., ). In parallel, the field of correlative light and electron microscopy has enabled the mapping of functional information onto high‐resolution ultrastructural electron microscopy data, by detecting fluorescent biomarkers in the context of cell structure (Kopek et al ., ; Bell et al ., ; Löschberger et al ., ; Johnson et al ., ; Bykov et al ., ; Mateos et al ., ; Wolff et al ., ). Integrated light and electron microscopy (ILEM) (Liv et al ., ) combines both microscopes in one device by placing a light microscope inside the vacuum chamber of an electron microscope.…”

Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells

“…Thicker regions, such as the cell interior surrounding the nucleus, can be investigated using several other complementary techniques, including sectioning-based approaches such as cryo-ultramicrotomy (i.e., CEMOVIS) 33,34 and cryo-focused ion beam milling 35,36 , or through scanning TEM-tomography approaches 37 . With the sectioning methods, additional experimental steps will be required so that the correlations to the fluorescent markers remain viable 38 . Another limitation of (cryo-)CLEM is the inability to precisely localize very small (<200-nm) molecules within a cell.…”