1.3 Microscopy

The study of cells and their structures, which are too small to be seen by the naked eye, is made possible through the technique of microscopy.

1. Light Microscopy

A light microscope, also known as a compound microscope, uses visible light and a system of two lenses to magnify specimens.

• Lenses:
  • Ocular Lens: Located in the eyepiece.
  • Objective Lens: Located in the revolving nosepiece.
• Working Principle:
  1. A light source illuminates the specimen.
  2. The objective lens forms an initial image that is real, inverted, and magnified.
  3. This first image acts as the object for the ocular lens (eyepiece).
  4. The eyepiece produces the final, virtual, and magnified image that the observer sees. The final image is inverted relative to the original specimen.

Magnification

Magnification is the ability of an optical instrument to increase the apparent size of an object. It is represented by the letter 'X'.

$\text{Magnification}=\frac{\text{Size of Image}}{\text{Actual Size of Object}}$

• Total Magnification: Calculated by multiplying the magnification of the objective lens by the magnification of the ocular lens.

$\text{Total Magnification}=\text{Objective Lens Power}\times \text{Ocular Lens Power}$

• Maximum Magnification: The maximum for a light microscope is typically 1500X (e.g., a 100X objective lens with a 15X ocular lens).

Resolution

Resolution (or resolving power) is a measure of the clarity of an image; it is the ability to distinguish between two points that are very close together.

• Higher resolution means the microscope can distinguish between points that are closer together (smaller minimum distance).
• Resolving Power (minimum resolvable distance):
  • Naked Eye: 0.1 mm
  • Light Microscope: 200 nm (0.2 μm)
  • Electron Microscope: 0.2 nm
• Increasing magnification does not increase resolution. Beyond a certain point, further magnification only produces a blurry image — this is called empty magnification.

2. Electron Microscopy

An electron microscope uses a beam of accelerated electrons as its source of illumination, allowing for significantly higher resolution and magnification than a light microscope.

• Working Principle:
  1. An electron gun produces a beam of electrons.
  2. Electromagnetic lenses (condenser lenses) focus the electron beam onto the specimen.
  3. The specimen must be an extremely thin section (20–100 nm).
  4. As the beam passes through, denser regions of the specimen scatter more electrons than lighter regions.
  5. The scattered electron pattern passes through an objective lens and then an ocular lens to create a highly magnified image on a fluorescent screen.

• Magnification: Can magnify specimens from 1 to 50 million times.
• Resolution: Has a resolving power of 0.2 nm — approximately 1000× better than a light microscope.

Types of Electron Microscopes

1. Transmission Electron Microscope (TEM): Electrons pass through an ultra-thin section of the specimen. Used to study the internal ultrastructure of cells and organelles.
2. Scanning Electron Microscope (SEM): Electrons are scanned across the surface of the specimen. Used to study the surface architecture (3D topography) of a specimen.

Comparison: Light vs. Electron Microscope

Ultrastructure of Animal and Plant Cells→