Microscope and its

classification
Dr Gurinderdeep Singh
Assistant Professor
Department Of Pharmaceutical Sciences and Drug Research
Punjabi University Patiala
• Microscopy can simply be
understood as the ‘use of
microscope’.
• Microscopy can be defined as the
scientific discipline of using
microscopes for getting a magnified
view of objects that can’t be viewed
by naked eyes.
• It is a very important tool in biology
and nanotechnology.
• In microbiology, it is one of the most
important tools used in observing
microbial cells.
• Medical sectors, pathology, histology,
molecular biology, and cytology are
in great debt of microscopy.
• There are three structural parts of the microscope i.e. head, base, and arm.
1.Head – This is also known as the body. It carries the optical parts in the upper
part of the microscope.
2.Base – It acts as microscopes support. It also carries microscopic illuminators.
3.Arms –
This is the part connecting the base and to the head and the eyepiece tube to the
base of the microscope.
It gives support to the head of the microscope and it is also used when carrying
the microscope.
Some high-quality microscopes have an articulated arm with more than one
joint allowing more movement of the microscopic head for better viewing.
• Optical parts of a microscope and their functions
• The optical parts of the microscope are used to view, magnify, and produce an image from a specimen placed on a slide.
These parts include:
1. Eyepiece – also known as the ocular. This is the part used to look through the microscope. Its found at the top of the
microscope. Its standard magnification is 10x with an optional eyepiece having magnifications from 5X to 30X.
2. Eyepiece tube – it’s the eyepiece holder. It carries the eyepiece just above the objective lens. In some microscopes such
as the binoculars, the eyepiece tube is flexible and can be rotated for maximum visualization, for variance in distance.
For monocular microscopes, they are none flexible.
3. Objective lenses – These are the major lenses used for specimen visualization. They have a magnification power of 40x-
100X. There are about 1- 4 objective lenses placed on one microscope, in that some are rare facing and others face
forward. Each lens has its own magnification power.
4. Nose piece – also known as the revolving turret. It holds the objective lenses. It is movable hence it cal revolve the
objective lenses depending on the magnification power of the lens.
5. The Adjustment knobs – These are knobs that are used to focus the microscope. There are two types of adjustment
knobs i.e fine adjustment knobs and coarse adjustment knobs.
6. Stage – This is the section in which the specimen is placed for viewing. They have stage clips that hold the specimen
slides in place. The most common stage is the mechanical stage, which allows the control of the slides by moving the
slides using the mechanical knobs on the stage instead of moving them manually.
•Abbe Condenser – this is a condenser specially designed for high-quality
microscopes, which makes the condenser to be movable and allows very high
magnification of above 400X. High-quality microscopes normally have a high
numerical aperture than objective lenses.
•The rack stop – It controls how far the stages should go preventing the
objective lens from getting too close to the specimen slide which may damage
the specimen. It is responsible for preventing the specimen slide from coming
too far up and hitting the objective lens.
• Phase Contrast Microscope
• Phase Contrast Microscope is an optical microscope that converts small phase shifts in light
into differences in light intensity developing more contrast in images that can be easily detected
by human eyes.
• When light passes through transparent specimens a small phase shift occurs which can’t be
detected by our eyes.
• Using phase plates, these small phase shifts are converted to changes in the amplitude of light.
• This change in amplitude can be observed as differences in image contrast.
• It can be used for observing living cells in their natural state without staining or fixing.
Transparent specimens and subcellular organelles can be clearly viewed with better contrast.
• Due to the difference in thickness and refractive index of different parts of a specimen, a small
phase shift in light rays occurs when the light passes through the specimens.
• This phase shift can be changed into differences in light intensity (brightness) which will
produce more contrast in the image.
• Phase Contrast Microscope Principle
• Light from the illuminator is focused on the specimen through the
condenser annulus.
• This light passes through different regions of the specimen having
different refractive indexes and thicknesses.
• The light rays that pass through an area of higher refractive index and
thickness, will experience larger phase retardation than those rays passing
through an area of lower refractive index and thickness.
• These phase shifts are undetectable to the normal human eye.
• An optical device like a phase plate converts these phase shifts into
brightness change which creates observable contrast differences in the
final image.
• Phase Contrast Microscope Parts
• It contains all the parts of a compound microscope, and additionally contains two optical parts, condenser annulus, and
phase plate, for phase contrast.
1. Condenser Annulus
• It is also called phase condenser or sub-stage annular diaphragm. It is an optical part that focuses a narrow hollow cone
of a light beam on a specimen to be observed.
• It is a black (light-absorbing) circular plate with a transparent annular ring/groove. The light passed through the annular
ring and fall on the specimen placed on the stage. In a microscope, it is placed below the condenser.
2. Phase Plate
• It is another optical part that selectively alters the phase and amplitude of light coming from the specimen. It is placed
above the objective rear focal plane.
• It is a circular transparent plate whose surface can be divided into two portions. The portion upon which the condenser
annulus is focused is termed the conjugate area. The remaining portion is collectively called a complementary area.
• The complementary area is coated with light retarding material like magnesium fluoride.
• The phase plate is of two types; a positive phase plate having a thinner conjugate area, and a negative phase plate
having a thicker conjugate area.
• Uses of Phase Contrast Microscope
• Observing living cells in its natural form
• Used in microbiology to observe protozoans, diatoms, planktons, cysts,
helminths and larvae.
• Used to study subcellular structures and cellular processes
• Used to study thin tissue slices
• Used to study lithographic pattern and latex dispersion, glass fragments and
crystals.
• Limitations of Phase Contrast Microscope
• Not ideal for thick specimen
• Halo effect and shade-off are common
• Condenser annulus limit the aperture, hence decrease resolution
• Dark Field Microscope

• Dark Field Microscope is a type of light microscope that uses only the
light scattered by the specimen for producing a bright image with a
darkfield around the specimen.

• It is a modified light microscope that uses an extra opaque disc below the
condenser lens which blocks the light coming from the source to reach the
objective lens.

• This microscope produces a higher resolution and better contrast than a

bright field microscope. There is no need to stain the specimens.
• Uses of Dark Field Microscope

• Used in microbiology to observe microbial motility, and spirochetes and other very thin bacteria.

• Used to study capsulated organisms

• Used in cytology to study internal organelles

• Used in computer mouse to allow the mouse to work over transparent medium

• It is coupled with hyperspectral imaging for characterizing nano particles

• Limitations of Dark Field Microscope

• Any contaminants like dust particles can scatter light giving false image

• Thin spreading of sample is mandatory

• Samples must be wet and moist and strongly illuminated

• Electron Microscope

• Electron Microscope is a microscope that uses accelerated electron, beams instead

of light rays, to illuminate the specimen and get the highly magnified image.

• In this microscope, glass lenses are replaced by electromagnets.

• Due to the very short wavelength of electrons, this microscope produces a very
high-resolution image with magnification up to 10,000,000X.

• Very high-quality images with very high contrast, revealing detailed structures are
produced.

• Specimen up to 0.2 nm can be clearly viewed using an electron microscope.

• Electron Microscope Principle
• An electron microscope uses accelerated electrons with a wavelength of about 100,000
times shorter than visible light to illuminate specimens and produce images.
• The electron gun, usually a heated tungsten or field emission filament, is used to generate a
stream of high voltage (100 – 1000 kV) electrons.
• These electrons are accelerated using an anode plate in a vacuum system and focused on
the specimen using aperture and electromagnetic lenses.
• The electron beam passes through the specimen and interacts with sample components.
Upon striking the specimen, the electrons are scattered.
• The degree of scattering depends on the refractive index or thickness of the specimen.
• The scattered electrons from the sample are collected and passed through objective and
ocular electromagnetic lenses.
• These scattered beams are detected and transformed into highly magnified images by the
magnetic lenses.
• Types of Electron Microscope
• There are different types of electron microscopes of which, TEM and SEM
are the most commonly used and important types. Some common types of
electron microscopes are;
1. Transmission Electron Microscope (TEM)
• Transmission Electron Microscope (TEM) is a type of electron
microscope that uses transmitted electrons to develop an enlarged image of
a specimen.
• In this system, very thin specimens, not more than 100 nm (about 200
times thinner than specimens used in the compound microscope), are used.
• Electrons are focused on the specimen using a condenser lens.
• The electrons interact with components of the specimen and get emitted out
from the sample.
• The emitted electrons are passed through objective and ocular
electromagnetic lenses and projected on a fluorescent screen.
• When the electrons hit the fluorescent screen, an enlarged image is
developed.
• It is the most commonly used electron microscope.
• It produces 2-D, black and white images with very high resolution and
magnification of 2 to 50,000X.
• Scanning Electron Microscope (SEM)
• Scanning Electron Microscope (SEM) is a type of
electron microscope that scans a specimen with a
high-energy beam of electrons in a raster scanning
pattern and develops a highly magnified 3-D image
of the specimen.
• The SEM uses emitted, backscattered, and diffracted
electrons for developing images reflecting the
characteristic morphological features of the
specimen.
• Although it has less magnification power than TEM,
the image will be of higher resolution and sharper.
• SEM contains some additional detector instruments
like back-scattered electron detectors, secondary
electron detectors, and X-ray detectors.