Abstract
The visualization of cellular ultrastructure over a wide range of volumes is becoming possible by increasingly powerful techniques grouped under the rubric “volume electron microscopy” or volume EM (vEM). Focused ion beam scanning electron microscopy (FIB-SEM) occupies a “Goldilocks zone” in vEM: iterative and automated cycles of milling and imaging allow the interrogation of microns-thick specimens in 3-D at resolutions of tens of nanometers or less. This bestows on FIB-SEM the unique ability to aid the accurate and precise study of architectures of virus-cell interactions. Here we give the virologist or cell biologist a primer on FIB-SEM imaging in the context of vEM and discuss practical aspects of a room temperature FIB-SEM experiment. In an in vitro study of SARS-CoV-2 infection, we show that accurate quantitation of viral densities and surface curvatures enabled by FIB-SEM imaging reveals SARS-CoV-2 viruses preferentially located at areas of plasma membrane that have positive mean curvatures.
Highlights
In the study of virus-cell interactions, high-resolution images that provide cellular context and ultrastructural information can provide unique and important insights complementary to those acquired by light microscopy
In HIV-1 infection, infected cells use a variety of membrane protrusions, including those involved in the virological synapse, to infect target cells [61,106], and we have recently shown that HIV-1 virions preferentially bud from infected cell plasma membrane patches with positive curvatures as measured by Focused ion beam scanning electron microscopy (FIB-scanning electron microscopy (SEM))
We have attempted to provide the virologist or cell biologist with a practical primer to focused ion beam (FIB)-SEM imaging as a volume Electron microscopy (EM) (vEM) approach as it pertains to capturing virus-cell interactions in 3-D and at high resolutions
Summary
In the study of virus-cell interactions, high-resolution images that provide cellular context and ultrastructural information can provide unique and important insights complementary to those acquired by light microscopy. Can interrogate significantly larger sample volumes at nanometer-level resolutions Of these vEM techniques, FIB-SEM has the resolving power in x, y, and z axes to accurately map and reconstruct features such as
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