TYPES OF
MICROSCOPES
Introduction·
The microscopic study of tissues (Histology) requires us
to visualize structures beyond the resolution of the naked
eye.
Microscopes are our primary tool. Major Classification
based on Illumination:
1. Light Microscopes (Optical Microscopes): Use visible
or ultraviolet light.
2. Electron Microscopes: Use a high-velocity beam of
electrons.
The Light Microscope (
Bright-Field)
This is the most commonly used microscope in student
labs.
It illuminates the specimen with white light that passes
through it.
The image is formed by the absorption of light by the
stained specimen, appearing against a bright background.
Components:
1. Illuminator (Light Source): Produces a beam of light.
2. Condenser Lens: Focuses the light onto the specimen.
3. Stage: Platform where the glass slide with the specimen
is placed.
4. Objective Lens: Gathers light from the specimen,
magnifies, and projects the image.
5. Ocular Lens (Eyepiece): Further magnifies the image
(usually 10x) and focuses it on the viewer's retina.
Resolving Power and Oil
Immersion
Resolving Power: The ability to distinguish between two
close points as separate.
For a bright-field microscope, it's about 0.2 μm.·
Magnification: It can magnify images up to 1000-1500
times.
Oil Immersion Objective (100x):
To increase the numerical aperture. · Light scatters when
it passes from the glass slide to air.
Phase-Contrast Microscope
Microscopes for Unstained & Living Cells·
Principle: Converts differences in refractive index and
thickness of cell structures into differences in light
intensity.
So different parts of cell bend light differently
Example; nucleus bend light more than cytoplasm
Use: To study living, unstained cells (e.g., cells in
culture).
So phase contrast microscope converts this bending
difference into brightness difference.
Dark-Field Microscope
Principle: Uses a special condenser that directs light
obliquely.
Only light scattered by the specimen enters the objective.
Image: Bright object against a black background. · Use:
To visualize unstained, transparent specimens (e.g., motile
bacteria like Treponema pallidum.
They are thin and cannot be seen easily in bright field.
Dark field makes it shine against the black background
Fluorescence Microscope
Principle: Specimens are stained with fluorescent dyes.
When illuminated with ultraviolet (UV) light, they emit
visible light.
Use: Highly specific and sensitive.
Used in immunocytochemistry (e.g., identifying specific
proteins using fluorescent antibodies).
We use antibody+ fluorescent dye. Antibody then binds to
protein and then the protein starts glowing
Example: Acridine orange stains DNA and RNA.
Confocal Microscope
It is advanced type of fluorescene microscope
Principle: Uses a laser and a pinhole aperture to eliminate
out-of-focus light.
Advantage: Produces sharp, high-resolution images of
thin optical sections through a thick specimen.
Can create 3D reconstructions.(Image: A fluorescence
image and a sharp confocal image of the same structure
Polarizing Microscope
Principle: Uses two filters (polarizer and analyzer) with
their axes perpendicular to each other.
Result: In the absence of a specimen, the field is dark.·
Birefringence (Double Refraction): · Structures with
highly ordered, asymmetric molecules (e.g., collagen,
microtubules, starch grains) rotate the polarized light and
appear bright against the dark background. · These are
called anisotropic
A histological section of bone or tendon viewed under
polarized light, showing bright birefringent collagen
fibers)
Electron Microscopy
Limitation of Light Microscopes: Resolution limited by
the wavelength of light.
Solution: Use a beam of electrons, which has a much
shorter wavelength (approx. 1/2000th of visible light).
This allows for much higher resolution (up to 2 nm) and
magnification (up to 120,000x).
Two Main Types:
1. Transmission Electron Microscope (TEM)
2. Scanning Electron Microscope (SEM)
Transmission Electron Microscope
(TEM)
Functions like a light microscope but
1. uses electrons instead of light,
2. and electromagnetic lenses instead of glass.
An electron gun produces a beam of electrons in a
vacuum.
The beam passes through an ultrathin section of the
specimen.
Denser structures scatter more electrons and appear
darker in the image.
The image is projected onto a fluorescent screen or
photographic plate
Use: To study detailed internal ultrastructure of cells
(organelles, membranes).
Scanning Electron Microscope
(SEM)
SEM: Seeing the Surface in 3D·
How it works
The specimen is coated with a thin layer of heavy metal
(e.g., gold).
A focused beam of electrons scans the surface. ·
Electrons reflected/ejected from the metal coat are
captured by a detector.
The signal is used to build a highly magnified 3-
dimensional image on a monitor.·
Use: To study the surface topography of specimens (e.g.,
the lining of a blood vessel, surface of a cell, a virus
particle).