What is resolution of a microscope? Comment on the principles of SEM and TEM. Describe the structure, working and applications of any one of the electron microscope in biology.

Resolution of a Microscope

Resolution (d) is defined as the minimum distance between two points that can be distinguished as separate entities. It is mathematically expressed as: d = 0.61λ/NA, where λ is the wavelength of the illuminating radiation and NA is the numerical aperture of the objective lens. Lower wavelengths and higher numerical apertures result in better resolution. Light microscopes are limited by the wavelength of visible light (~400-700 nm), restricting their resolution to approximately 200 nm. Electron microscopes, using electron beams with much shorter wavelengths, achieve resolutions down to the angstrom level (0.1 nm), allowing visualization of individual atoms.

Scanning Electron Microscopy (SEM) vs. Transmission Electron Microscopy (TEM)

Both SEM and TEM utilize electron beams, but differ significantly in their principles, sample preparation, and applications.

Feature	Scanning Electron Microscopy (SEM)	Transmission Electron Microscopy (TEM)
Principle	Scans a focused electron beam over the surface of a sample. Detects secondary electrons emitted from the surface.	Transmits a beam of electrons *through* an ultra-thin specimen. Forms an image based on electron transmission.
Sample Preparation	Samples are coated with a thin layer of conductive material (e.g., gold, platinum) to prevent charge buildup. Generally requires less extensive preparation.	Requires ultra-thin sections (typically 60-100 nm) prepared using an ultramicrotome. Often involves fixation, embedding, and staining with heavy metals.
Image Type	Provides detailed 3D images of the sample surface.	Provides 2D projections of the internal structure of the sample.
Resolution	Typically 1-20 nm.	Typically 0.2-0.5 nm.
Applications	Surface morphology, materials science, forensic science.	Internal ultrastructure of cells, viruses, and materials.

Transmission Electron Microscopy (TEM): Structure, Working, and Applications

Structure

A TEM consists of several key components:

• Electron Gun: Generates a beam of electrons, typically using a tungsten filament or LaB6 crystal.
• Condenser Lenses: Focus the electron beam onto the specimen.
• Specimen Stage: Holds the ultra-thin specimen.
• Objective Lens: Forms the initial magnified image.
• Projector Lenses: Further magnify the image.
• Fluorescent Screen/Detector: Detects the transmitted electrons and displays the image.
• Vacuum System: Maintains a high vacuum within the column to prevent electron scattering.

Working

The process begins with preparing an ultra-thin section of the sample. The specimen is then placed on a copper grid and inserted into the TEM column. The electron gun emits a beam of electrons, which is focused by the condenser lenses onto the specimen. As the electrons pass through the specimen, some are scattered, while others are transmitted. The objective lens forms an initial magnified image of the transmitted electrons. This image is further magnified by the projector lenses and projected onto a fluorescent screen or detected by an electronic detector, creating a visible image. Contrast is achieved by varying the electron density of different parts of the specimen, often enhanced by staining with heavy metals like uranium or lead.

Applications

• Cell Biology: Studying the ultrastructure of organelles (mitochondria, endoplasmic reticulum, Golgi apparatus), viruses, and cellular components.
• Virology: Visualizing the morphology and structure of viruses.
• Materials Science: Analyzing the microstructure of materials, including metals, polymers, and ceramics.
• Nanotechnology: Characterizing nanoparticles and nanomaterials.
• Pathology: Identifying pathogens and diagnosing diseases based on cellular abnormalities.