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Microscopy in Virology

The coronavirus SARS-CoV-2, causing the Covid-19 disease effects our world in all aspects. Research to find immunization and treatment methods, in other words to fight this virus, gained highest priority in 2020. Microscopes play a significant role in this kind of research. To understand the underlaying principles of receptor binding, genome release, replication, assembly, and virus budding, as well as the response of our immune system, different methodologies and microscopes can be used. This article summarizes why microscopy is an important tool in virology and infection biology and gives examples for different microscopy technologies and their applications in these research fields.

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Broad range of microscopy applications for virology

The application range of light microscopy for virologists is broad. Most of them are based on fluorescence. Fluorescence microscopy can be roughly subdivided into immunofluorescence and the utilization of fluorescent proteins. Immunofluorescence utilizes fixed cells or tissues to stain the protein of interest with fluorescently marked antibodies, while fluorescent proteins can also be utilized for live-cell imaging.

Microscopy methods used in virology range from widefield microscopy, through confocal microscopy, to super-resolution microscopy. Moreover, there is also the applicability of Laser Microdissection.

The following references will give you an overview of prominent microscopy methods and their application in virology:

Influence of the cellular lipid metabolism on coronavirus replication

Müller et al. studied the influence of the cellular lipid metabolism on coronavirus replication. They infected Huh-7 cells with the coronavirus HCov-229E and stained these cells with antibodies against dsRNA, the coronavirus N protein, and the coronavirus NSP8 protein. Moreover, they marked lysophospholipids with the help of the BODIPY phospholipid PC-A2. Imaging was then executed with a TCS SP5 confocal microscope equipped with a 63x Plan-Apochromat objective. The researchers were interested in colocalization events of dsRNA and PC-A2.

The cytosolic phospholipase A2α (cPLA2α) produces phospholipids. When they treated the cells with an inhibitor of cPLA2α, they could detect a significant effect on viral RNA and protein accumulation in human coronavirus cells. In more detail, they observed that RTC (viral replication/transcription complex) co-localized with lysophospholipid containing membrane structures. For this reason, they conclude that cPLA2α could be a target for an antiviral drug. https://jvi.asm.org/content/92/4/e01463-17#sec-9

Viruses have their own RNA or DNA. Due to this fact, they can be detected by fluorescence in situ hybridization (FISH) where a fluorescent probe is used to target viral nucleotides.

Bacterial infection in the mouse mucosal barrier

Jarret et al. did not investigate viral but rather bacterial infection in the mouse mucosal barrier. By using single-molecule fluorescence in situ mRNA hybridization (smFISH; THUNDER Imager 3D Live Cell) they discovered that intestinal neurons produce the cytokine IL-18. With it, they could show that neuron derived IL-18 signaling plays a major role in intestinal immunity which was not yet discovered.
https://www.cell.com/cell/fulltext/S0092-8674(19)31378-9

Monitor markers for immunosuppressive mechanisms

Immunosuppression can be studied with the help of viral infected mouse models. Andrew Beppu used large multi-channel tile scans of mouse lungs to monitor markers for immunosuppressive mechanisms. The mice were infected with the influenza virus and then investigated for regression of lung basal-like structures (THUNDER Imager 3D Cell Culture).

DENV induced gene expression effects

Sim et al. studied Dengue virus (DENV) induced gene expression effects in the salivary gland of the yellow fever mosquito (Aedes aegypti). The authors found out that upon DENV infection, the transcriptome of the mosquito salivary gland was changed. By using microarrays for gene expression analysis, they could show for the first time that some genes were expressed which have influence on the mosquito’s host-seeking and probing behavior. They used immunofluorescence microscopy to identify regions in the mosquito’s chemosensory organs where the virus was abundant (see Fig. 3).
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1002631#s4

The morphology of Hendra virus infected cells

Monaghan et al. characterized the morphology of Hendra virus infected cells with the help of microscopy. Confocal images (SP5) revealed the intracellular viral protein distribution, whereas super-resolution microscopy (SR-GSD) even gave a closer look into the protein distribution inside virions. 
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4254186/

Cellular RNA helicase binding to La Crosse virus (LACV) nucleocapsids

Super-resolution microscopy was utilized by Weber et al. to study cellular RNA helicase binding to La Crosse virus (LACV) nucleocapsids (SR-GSD). For this purpose, immunofluorescence was applied to stain the viral LACV N protein and cellular RNA helicase RIG-I in infected A549 cells. Super-resolution microscopy supported biochemistry data that RIG-I binds the nucleocapsids via the 5’ppp dsRNA panhandle.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5515363/

Natural killer (NK) cells

Maze and Orange investigated natural killer (NK) cells which are important counterplayers of viruses. They belong to the innate immune system and surveil virally infected and tumorigenic cells. Directed secretion of specialized secretory lysosomes via the immunological synapse kills the virally infected cells. STED nanoscopy (TCS SP8 STED) was used to decipher the interaction of lytic granules with the cytoskeleton of the NK cells.
https://www.leica-microsystems.com/science-lab/visualization-of-the-natural-killer-cell-immune-synapse-by-super-resolution-nanoscopy/

Image the immunological synapse with STED nanoscopy

Another publication by Zheng et al. describes the technical procedure which can be used to image the immunological synapse with STED nanoscopy (TCS SP8 STED).
https://www.jove.com/video/52502/super-resolution-imaging-natural-killer-cell-immunological-synapse-on

Shed light on parasitology problems with STED nanoscopy

In addition to virology and immunology, STED nanoscopy can also shed light on parasitology problems, such as merozoite invasion in erythrocytes. 3D STED (TCS SP8 STED) reveals spatial information of the protein components which are involved in Plasmodium infection. 
https://www.leica-microsystems.com/science-lab/observing-malaria-infection-at-the-right-spot-in-the-human-host/

Influence of coronaviruses on cellular NF-κB signaling and chromatin landscape

Poppe et al. investigated the influence of coronaviruses on cellular NF-κB signaling and chromatin landscape. With the help of Laser Microdissection (LMD6000), they isolated cells expressing the coronavirus N protein and extracted their entire RNA. By utilizing RT-qPCR and microarray analysis, they found out which mRNAs were over- or under-represented in the cells. Furthermore, they used immunofluorescence of the coronavirus N protein to monitor viral infection and its spread in A546 cells (DMIRE2, DMi8). 
https://journals.plos.org/plospathogens/article?id=10.1371/journal.ppat.1006286#sec002)

Brightfield microscopy plays a direct, but only minor role in virology, because fluorescence microscopy fulfills better the demands of researchers. Nevertheless, there are opportunities for brightfield microscopy when animal tissues are investigated. For example, researchers can check tissues for morphology changes after viral infection, etc.

In addition, brightfield microscopy is used in cell culture labs to check the health and growth status of cells (see Fig. 5) which are already infected or will be at a later time (DM IL, DMi1, PAULA).

This list of microscopy methods is not meant to be comprehensive. There are other techniques which are also used to visualize viruses, but are not in the scope of this article. For example, electron microscopy (EM) can resolve virus particles. Single molecule detection (TCS SP8 SMD) and Fluorescence Lifetime Imaging (FLIM) (STELLARIS 8 FALCON), as well as multiphoton microscopy (SP8 DIVE) are additional methods, which are suitable for virology (see references).

Fig. 6: Sample preparation for electron microscopy. Image taken from https://www.youtube.com/watch?v=dtKT0FUKKxA at EMBL, Heidelberg.

SARS-CoV and SARS-CoV-2 were able to infect enterocytes

Lamers et al. investigated, if the coronavirus SARS-CoV-2 infects not only the respiratory system, but also the human gut. By using confocal microscopy (SP8) and electron microscopy (EM sample preparation: EM UC7) with human small intestine organoids, they could show that SARS-CoV and SARS-CoV-2 were able to infect enterocytes. https://science.sciencemag.org/content/early/2020/04/30/science.abc1669

To find out more about electron microscopy, please refer to the examples given in the references below, as well as articles dedicated to the topic.

References

Confocal Microscopy in Virology

Single Viruses on the Fluorescence Microscope: Imaging Molecular Mobility, Interactions and Structure Sheds New Light on Viral Replication.

Visualizing Oncolytic Virus-Host Interactions in Live Mice Using Intravital Microscopy

Inhibition of Cytosolic Phospholipase A2α Impairs an Early Step of Coronavirus Replication in Cell Culture

Detailed morphological characterization of Hendra virus infection of different cell types using super-resolution and conventional imaging

Characterization of Virus-Specific Vesicles Assembled by West Nile Virus Non-Structural Proteins

Host Sphingomyelin Increases West Nile Virus Infection in Vivo

A Vaccine Based on a Modified Vaccinia Virus Ankara Vector Expressing Zika Virus Structural Proteins Controls Zika Virus Replication in Mice

Targeting Host Metabolism by Inhibition of acetyl-Coenzyme A Carboxylase Reduces Flavivirus Infection in Mouse Models

Fluorescence Lifetime Imaging (FLIM) in Virology

Quantitative FRET-FLIM-BlaM to Assess the Extent of HIV-1 Fusion in Live Cells

Single Molecule Detection (SMD) in Virology

A Dynamic Three-Step Mechanism Drives the HIV-1 Pre-Fusion Reaction

Toremifene Interacts With and Destabilizes the Ebola Virus Glycoprotein

Dynamin-2 Stabilizes the HIV-1 Fusion Pore With a Low Oligomeric State

Astrocytes Resist HIV-1 Fusion but Engulf Infected Macrophage Material

Multiphoton Microscopy in Virology

Single-cell glycolytic activity regulates membrane tension and HIV-1 fusion

Widefield Microscopy in Virology

Dengue Virus Infection of the Aedes aegypti Salivary Gland and Chemosensory Apparatus Induces Genes that Modulate Infection and Blood-Feeding Behavior

Super-Resolution Microscopy in Virology

Detailed morphological characterization of Hendra virus infection of different cell types using super-resolution and conventional imaging

JC Virus Inclusions in Progressive Multifocal Leukoencephalopathy: Scaffolding Promyelocytic Leukemia Nuclear Bodies Grow With Cell Cycle Transition Through an S-to-G2–Like State in Enlarging Oligodendrocyte Nuclei

Incoming RNA virus nucleocapsids containing a 5′-triphosphorylated genome activate RIG-I and anti-viral signaling

Single HIV-1 Imaging Reveals Progression of Infection Through CA-Dependent Steps of Docking at the Nuclear Pore, Uncoating, and Nuclear Transport

Laser Microdissection in Virology

The NF-κB-dependent and -independent transcriptome and chromatin landscapes of human coronavirus 229E-infected cells

Electron Microscopy in Virology

Microwave Assisted Rapid Diagnosis of Plant Virus Diseases by Transmission Electron Microscopy

Cryo-electron Microscopy of Tubular Arrays of HIV-1 Gag Resolves Structures Essential for Immature Virus Assembly

Rapid Immunohistochemical Diagnosis of Tobacco Mosaic Virus Disease by Microwave-assisted Plant Sample Preparation

SARS-CoV-2 productively infects human gut enterocytes

EMBL scientists using electron microscopy to look at coronavirus infected cells

Other Infectious Agents

Observing Malaria Infection at the Right Spot in the Human Host

Inflammation

Chronic Inflammation Under the Microscope

Visualization of the Natural Killer Cell Immune Synapse by Super-Resolution Nanoscopy

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

Cell Culture

Introduction to Mammalian Cell Culture - Morphology and Cell Types & Organization

Resolution

Super-Resolution – On a Heuristic Point of View About the Resolution of a Light Microscope