Microbiological

techniques: Microscopy,
principles and types of
microscopy; optical, TEM
and SEM

By – Dr. Jagnoor Singh

 History of microscope
• In 1590 F.H Janssen & Z. Janssen constructed the first simple compound light microscope -10x to 30x .
• In 1665 Robert Hooke developed a first laboratory compound microscope.
• Later, Kepler and Galileo developed a modern class room microscope.
• In 1672 Anton Von Leeuwenhoek developed a first simple microscope with a magnification of 200x – 300x.
• In 1674, Anton was the first to see and describe bacteria, yeast, plants, and life in a drop of water- He is called
as Father of microscopy.
• The term microscope was coined by Faber in 1623.
• In the early 1930’s the first electron beam microscopes were developed which were a breakthrough in
technology as they increased the magnification from about 1000x or so up to 250,000x or more.
History of the
Microscope
• 1590 –first compound
microscope
History of the
Microscope
• 1655 – Robert Hooke used a
compound microscope to
observe pores in cork.

• He called them “cells”

Microscopy
• Microscope is used to view objects or specimens that are too small to be seen with
just the human eye.
• It is the technique of making very small things visible to the unaided human eye. It is
the study of the principles and techniques of visualization of microorganisms
through the microscope
• Definition-A microscope is a high precision optical instrument that uses a lens or a
combination of lenses to produce highly magnified images of small specimens or
objects especially when they are too small to be seen by the naked (unaided) eye.
• A light source is used (either by mirrors or lamps) to make it easier to see the subject
matter.
 Principle

Microscopy is used to get a magnified image, in which structures may be

resolved, which could not be resolved with the help of an unaided eye.

Magnification
• It is the ratio of the size of an object seen under microscope to the
actual size observed with unaided eye.
• The total magnification of microscope is calculated by multiplying the
magnifying power of the objective lens by that of eye piece.
 Magnification

• Your microscope has 3 magnifications: Scanning, Low and High.

Each objective will have written the magnification.
In addition to this, the ocular lens (eyepiece) has a magnification.
The total magnification is the ocular x objective.
Numerical Aperture
• The numerical aperture of a lens is the ratio of the diameter of the lens to its focal length.

• The light-gathering ability of a microscope objective is quantitatively expressed in terms of the

numerical aperture.

Diameter of the lens

Numerical Aperture
• The more light (higher NA) the better the resolving power of the lens and better the
resolution

The typical N.A. for the following are;

• 4x=0.10,
• 10x=0.25
• 40x=0.65
• 100x=1.25.
 Resolving power/limit of resolution

• It is the ability to differentiate two close points as separate.

• The resolving power of human eye is 0.25 mm
• The light microscope can separate dots that are 0.25µm apart.
• The electron microscope can separate dots that are 0.5nm
apart.
 Working distance

• It is the distance between the objective and the objective slide.

• The working distance decreases with increasing magnification.
Lenses and the Bending of Light

• light is refracted (bent) when passing from one medium to another.

Refractive index
• A measure of how greatly a substance slows the velocity of light

Direction and magnitude of bending is determined by the refractive indexes of the two
media forming the interface
Lenses

• Focus light rays at a specific place called the focal point

• Focal length is the distance between centre of lens and focal
point.
• The strength of lens related to focal length
• Short focal length more magnification
Introduction to microscopes
• The image produced is magnified by a combination of the objective lens and the
eyepiece lens.
• Usually, the eyepiece lens gives a x10 magnification.
• Three objective lenses are usually used: x10, x40 and x100
• The x100 lens is usually an oil-immersion lens - you need to view the sample through
a drop of oil.
For 100x magnification, apply immersion oil on the slide and switch to the oil immersion lens. Use fine
focus for sharp detailing.
Types of
Microscopes
Compound Light Microscope
• 1st type of microscope, most
widely used
• light passes through 2 lenses
• Can magnify up to 1000x
Types of
Microscopes
Electron Microscope
• Used to observe VERY small
objects: viruses, DNA, parts of
cells
• Uses beams of electrons rather
than light
• Much more powerful
Types of Electron
Microscopes
• Transmission Electron
Microscope (TEM)
• Can magnify up to 250,000x
Types of Electron
Microscopes
• Scanning Electron Microscope
(SEM)
• Can magnify up to 100,000x
Imaging Techniques

Technique Image Formed By

Optical Microscopy Light Rays
Confocal Microscopy Coherent Light Source (Laser)

Transmission
Electrons
Electron Microscopy (TEM)

Scanning Electron Microscopy (SEM) Electrons

Atomic Force & Scanning Tunneling Microscopies

Molecular Mechanical Probes
(AFM/STM)
Compound Microscope
• Common type of microscope.
• High power microscope- The magnification (power) 40x to 1000x.
• Compound refers to the fact that in order to enlarge an image - a
single light path passes through a series of lenses in a line where
each lens magnifies the image over the previous one.
• In the standard form – 2 lenses
• An objective lens (closest to the object or specimen)
• An eyepiece lens (closest to the observers’ eye)
Compound Microscope
• Uses light to illuminate the specimen
• The objective lens usually consists of three or four lenses.
• The most used light method is trans-illumination.
• At 400x much detail can be seen at the cellular level of
biological specimens.
• Applications: Learn about cells and microorganisms in both
medical and science field.
Objective lenses
• One of the most important parts of a compound microscope, as they
are the lenses closest to the specimen.
• A standard microscope has three, four, or five objective lenses that
range in power from 4X to 100X.
• Objectives vary in power from 1x to 160x in compound microscopes
but the most common power range is from 4x to 100x.
• Most compound microscopes have three or four (occasionally five)
objectives usually of 4x, 10x, 40x, and 100x (oil immersion) which
revolve on a nosepiece (turret) to give different magnifying powers.
Ocular Lens or Eye piece
• The eyepiece consists of a series of lenses mounted in a tube (barrel) at the upper
end of the microscope.
• Its basic function is to look at the focused, magnified image projected by the
objective lens and magnify that image a second time before your eye looks at the
image of the specimen.
• The eyepieces are usually 10x but also come in 5x, 12.5x, 15x, and 20x. The “x” refers
to the amount of magnification (power) that this lens adds as a multiplier to the
magnification of the objective.
• For special applications, eyepieces can have scales, pointers, crosshairs, markers,
etc. on them.
• The eyepoint is the location (or position) of the eye from the eyepiece which allows
for the best possible viewing of the image.
Other parts
• Diopter Adjustment: Useful as a means to change focus on one eyepiece so as to
correct for any difference in vision between your two eyes.
• Body tube (Head): The body tube connects the eyepiece to the objective lenses.
• Arm: The arm connects the body tube to the base of the microscope.
• Coarse adjustment: Brings the specimen into general focus.
• Fine adjustment: Fine tunes the focus and increases the detail of the specimen.
• Nosepiece: A rotating turret that houses the objective lenses. The viewer spins the
nosepiece to select different objective lenses.
• Specimen or slide: The specimen is the object being examined. Most specimens
are mounted on slides, flat rectangles of thin glass.
 Other parts
• Stage: The flat platform where the slide is placed.
• Stage clips: Metal clips that hold the slide in place.
• Stage height adjustment (Stage Control): These knobs move the stage left and right or up
and down.
• Aperture: The hole in the middle of the stage that allows light from the illuminator to reach
the specimen.
• On/off switch: This switch on the base of the microscope turns the illuminator off and on.
• Illumination: The light source for a microscope. Illumination is the application of light onto
an object or specimen in a microscope.
• Diaphragm: Adjusts the amount of light that reaches the specimen.
• Condenser: Gathers and focuses light from the illuminator onto the specimen being viewed.
• Base: The base supports the microscope and it’s where illuminator is located.
Using the microscope
• General Procedures

• The working area- Make sure all backpacks and materials are out of the
aisles and off the tops of desks, specially any pointed objects like
needles.

• Power cord - Plug your microscope in to the outlet.

Store with cord wrapped around microscope.

• Carrying the equipment- Carry by the base and arm with both hands.
Focusing Specimens

• Begin with the lowest power objective lens to locate the

specimen.
• Use the Coarse Knob to focus and then the fine adjustment knob
until clear, image may be small at this magnification.
• Modify the diaphragm or iris to adjust the amount of light,
improving the visibility and contrast of the specimen.
• Rotate the nosepiece to switch to a higher power objective lens.
Use ONLY the fine adjustment knob to refine the focus.
1. When focusing a specimen, you should always start with the
___________________ objective.

2. When using the high power objective, only the ___________

knob should be used.

3. The type of microscope used in most science classes is the

_________________ microscope

4. What part of the microscope can adjust the amount of light that hits
the slide? ______________________________
5. You should carry the microscope by the ________ and the __________.

6. The objectives are attached to what part of the microscope (it can be
rotated to click the lenses into place):
_______________ ________________

7. You should always store you microscope with the ________________

objective in place.

8. A microscope has an ocular objective of 10x and a high power objective of

50x. What is this microscope's total magnification? ____________
The Bright-Field Microscope

• Definition: A standard microscopy technique where light is transmitted through

the sample, and the image is observed directly.
• Key Components:
• Light source: Illuminates the specimen.
• Condenser lens: Focuses light on the specimen.
• Objective lenses: Magnify the image (typically 4x, 10x, 40x, 100x).
• Ocular lens (eyepiece): Further magnifies the image for viewing.

Brightfield microscopy, also known as a compound light microscopy, uses

light to illuminate a sample and create an image.
Advantages Disadvantages

• Simplicity: Easy to use and • Low contrast in unstained

requires minimal training. specimens: Difficult to see details
• Cost-effectiveness: Relatively without staining.
inexpensive compared to more • Limited resolution: Cannot resolve
advanced techniques. structures smaller than about 200
• Effective for stained specimens: nm.
Provides good contrast for • Not ideal for live specimens:
specimens that can be stained. Staining often required, which can
kill or alter live samples.
Applications of Bright Field Microscopy
• Pathology: Examination of tissue sections for disease diagnosis.
Applications of Bright Field Microscopy
Microbiology: Observation of bacteria and fungi.

A B
Applications of Bright Field Microscopy
• Cytology: Study of cell morphology and structure

A B
Dark Field Microscopy
• A microscopy technique that enhances contrast by illuminating
the specimen with light that does not enter the objective lens
directly.
• Light is directed from the side and scattered by the specimen.
• Bright image on dark background: Only scattered light enters the
objective lens, making the specimen appear bright against a dark
background.
Dark Field Microscopy
Advantages Disadvantages
1. Enhanced contrast for unstained • Requires special equipment: Needs
specimens: Excellent for observing a specialized condenser and light
transparent or colorless stop.
specimens. • Sensitivity to dust and impurities:
2. Suitable for live specimens: Allows Any debris or imperfections can
observation of live microorganisms appear prominently in the image.
and cells without staining. • Limited quantitative analysis:
3. Detects small structures: Effective Difficult to measure specific
for observing fine details and small properties of the specimen.
particles.
Applications
Treponema pallidum
Applications

Material Science:
Detection of small
defects and particles in
materials.
Applications
• Examining live blood cells
Fluorescence Microscopy
• A type of optical microscopy that uses fluorescence to generate an
image.

• Fluorescent molecules (fluorophores) absorb light at a specific

wavelength (excitation) and emit light at a longer wavelength (emission)
• Excitation light is filtered through an excitation filter
• Emitted light is separated from the excitation light by a dichroic mirror
and emission filter
• Only the emitted light is detected, producing a high-contrast image
Applications
• Cell biology: imaging organelles, proteins, and other cellular
structures
• Molecular biology: tracking gene expression, protein interactions
• Medical diagnostics: identifying pathogens, cancer cells
• Neuroscience: mapping neural circuits, studying synapses
• Environmental science: studying microbial communities, biofilms
Fluorescence Advantages Limitations

Microscopy • High specificity: Ability to • Photobleaching:

target specific molecules Fluorophores can degrade
• High contrast: Bright upon prolonged exposure to
fluorescence against a dark light
background
• Phototoxicity: High-intensity
• Live-cell imaging: Can be light can damage live cells
used to observe live
specimens • Background fluorescence:
Autofluorescence can
• Multi-color imaging: Use of interfere with signal
multiple fluorophores to
visualize different • Limited resolution:
structures simultaneously Diffraction limit restricts
resolution to about 200 nm.
Fluorescence
Microscopy
• Example: DAPI stained to show the
stage of cell division.
• Top: (Interphase) Only the nucleus
is visible
• Bottom: (Mitosis) chromosomes
lined up at centre of cell.
Rabies Virus detection using Fluorescent Antibody Testing
Phase Contrast Microscopy
• Unstained preparation such as saline wet mount is often used to
demonstrate living microorganisms and their motility. Visualization of
their morphology and internal structures is difficult because they don’t
have colour of their own and contrast poorly with the background.
• Phase contrast microscope is used to create contrast between the
organism, its structures and the background, thus making its visibility
quite clear.

Key Components
• Phase annular diaphragm
• Phase plate in the objective lens
• Condenser with phase rings
Phase Contrast Microscopy
Advantages Disadvantages
• Visualize transparent • Halo artifacts around
specimens without staining specimen structures
• Observe live cells and dynamic • Limited quantitative analysis
processes
• Enhances contrast of • Requires specialized
structures with similar equipment
refractive indices
Applications
• Cell biology: studying live cells, organelles, and motility
• Microbiology: observing bacteria and protozoa
• Embryology: monitoring embryo development
Tobacco mosaic virus-infected tobacco leaf Thin section of the snake skin
HeLa Cell Culture
Thank You