Viruses are microscopic entities, too small to be seen without specialized tools. Understanding their structure, infection mechanisms, and replication requires advanced imaging technology. This article explores the microscopic techniques developed to visualize these tiny biological structures.
Limits of Light Microscopy
Traditional light microscopes focus visible light through a sample, effective for observing cells and bacteria (micrometers in size). However, viruses are significantly smaller, generally 20 to 400 nanometers. The fundamental limitation is the diffraction limit: an object cannot be resolved if smaller than about half the wavelength of light used. Since visible light wavelengths range from 400 to 700 nanometers, viruses fall well below this threshold, preventing clear images of individual particles.
The Power of Electron Beams
Overcoming light microscopy’s limitations led to electron microscopy. These instruments use a beam of electrons instead of photons. Electrons have a much shorter wavelength than visible light; when accelerated to high voltages, their de Broglie wavelength can be thousands of times shorter. This enables significantly higher resolution, allowing electron microscopes to resolve details down to the atomic scale, making them suitable for visualizing viruses. They employ electromagnetic lenses, not glass, to focus and manipulate the electron beam, directing it through or onto a sample to generate an image.
Transmission Electron Microscopy
Transmission Electron Microscopy (TEM) examines the internal structures of viruses. An electron beam passes through a thin sample. Electrons interact with the sample, some scattering while others pass unimpeded. This creates variations in the beam, forming a high-resolution image on a screen or digital detector. TEM reveals details like the viral capsid, genetic material arrangement, and internal enzymes. Sample preparation involves fixing the virus, embedding it in resin, slicing ultrathin sections (50-100 nanometers), and staining with heavy metals like uranium or lead for contrast.
TEM has characterized the morphology of many viruses, providing insights into their structural organization. It helped determine the shapes of bacteriophages, influenza viruses, and adenoviruses. This method is useful for understanding how viruses assemble within host cells or interact with cellular components. The images produced are two-dimensional projections, offering a cross-sectional view.
Scanning Electron Microscopy
Scanning Electron Microscopy (SEM) creates three-dimensional images of viral surfaces. Unlike TEM, SEM scans a focused electron beam across the sample’s surface. When the primary beam strikes, it dislodges secondary electrons from surface atoms. Detectors collect these electrons, and their varying numbers, based on topography, construct an image. This process reveals external features like shape, texture, and surface protein arrangement, which are important for viral attachment to host cells.
For SEM sample preparation, viruses are fixed and coated with a thin layer of an electrically conductive material, such as gold or platinum. This coating prevents charge buildup and enhances secondary electron emission, leading to clearer images. SEM is useful for observing how viruses bud from infected cells or cluster. It offers a view of viral populations on a surface, complementing TEM’s internal details.
Cryo-Electron Microscopy
Cryo-Electron Microscopy (Cryo-EM) allows scientists to visualize viruses in a near-native, hydrated state. This technique rapidly freezes a thin layer of virus solution (less than 100 nanometers thick) in liquid ethane. This rapid freezing, called vitrification, prevents ice crystal formation that could damage viral structures. The vitrified sample is then examined at cryogenic temperatures. Since no chemical fixatives or heavy metal stains are used, Cryo-EM avoids artifacts from traditional sample preparation.
Cryo-EM captures multiple two-dimensional images of identical virus particles from different angles. Computational algorithms combine these images to reconstruct a high-resolution three-dimensional model. This approach has transformed structural biology, enabling atomic-resolution structures for many viruses, including Zika virus and SARS-CoV-2. Visualizing viruses in their natural conformation provides insights into their assembly, antibody interaction, and infection mechanisms, impacting vaccine and antiviral drug development.