Concepts in Light Microscopy of Viruses

Abstract

Viruses threaten humans, livestock, and plants, and are difficult to combat. Imaging of viruses by light microscopy is key to uncover the nature of known and emerging viruses in the quest for finding new ways to treat viral disease and deepening the understanding of virus–host interactions. Here, we provide an overview of recent technology for imaging cells and viruses by light microscopy, in particular fluorescence microscopy in static and live-cell modes. The review lays out guidelines for how novel fluorescent chemical probes and proteins can be used in light microscopy to illuminate cells, and how they can be used to study virus infections. We discuss advantages and opportunities of confocal and multi-photon microscopy, selective plane illumination microscopy, and super-resolution microscopy. We emphasize the prevalent concepts in image processing and data analyses, and provide an outlook into label-free digital holographic microscopy for virus research.

Formation of adenovirus DNA replication compartments

“…However, the role of many of these viral and cellular proteins in virus replication and/or control of cellular activities has not been determined, mainly because microscopy techniques and the limited number of molecules analyzed simultaneously preclude a detailed dissection of the molecular mechanisms at play in the AdRCs. Novel single‐particle, super‐resolution imaging technologies in live cells (see review by Pied & Wodrich in this issue) and analyses of the factors associated with replicating viral DNA, using isolation of proteins on nascent DNA coupled with mass spectrometry (see review by Charman et al in this issue ) or the isolation of AdRCs from subnuclear fractions that support de novo viral DNA and RNA synthesis and the splicing of viral late mRNA , will help to better define the dynamic organization, composition, and function of AdRCs, and the underlying regulatory mechanisms.…”

The viral replication organelles within cells studied by electron microscopy

“…Furthermore, new advances in fluorescent probes and LM methods are providing new insights into the cell biology of viruses. Very interesting recent reviews deal with light microscopy of viral infections (Alonas et al, 2016;Dirk et al, 2016;Hanne et al, 2016;Witte et al, 2018). However, microscopy of fixed cells provides static views of complex, dynamic events.…”

Label-free digital holo-tomographic microscopy reveals virus-induced cytopathic effects in live cells

“…Furthermore, it can be used to acquire two new dimensions of cell data, RI and RIG, which cannot be readily assessed by other imaging modalities. The methodology is robust, and overcomes a series of technical limitations in live cell pathogen imaging, including the labeling of cellular and viral entities by chemical or geneticallyencoded fluorophores, and toxicity ensuing imaging (for recent reviews, see (38,39)). The non-invasiveness and low laser power required for DHTM underscore the suitability of DHTM for long-term live imaging.…”