MICROSCOPY BY INSHA JAN

Seminar Presentation
on
Microscopy
Department of Microbiology
SKIMS SOURA

By definition a microscope
 An optical instrument consisting of a lens or combination of lenses to
produce magnified images of small objects especially which are too small
to be seen by naked eye.
Microscope
Derived from two Greek Words
 Micron means “Small”
 Scope means “Aim”
INTRODUCTION HISTORICAL BACKGROUND PROPERTIES OF MICROSCOPE
PARTS OF MICROSCOPE TYPES OF MICROSCOPE

 Magnification or magnifying power
• is the degree of increase in size of optical image over the actual size of object
being viewed.
• Total magnification of a field is the product of the magnification of objective
lens and ocular lens:
• Scanning field ( 40x)
• Low power field (100X)
• High power field (400x) and
• Oil immersion field (1000X)

 Resolution power:
• Ability to produce separate images of closely placed objects so that they can be
distinguished as two separate entities. The resolution
• Unaided human eye is about 0.2 mm (200 μm).
• Light microscope is about 0.2 μm.
• Electron microscope is about 0.5 nm (0.2 μm).
• Resolution depends on refractive index of the medium.
• Oil has a higher refractive index than air; hence, use of oil enhances the
resolution power of a microscope.

 Good contrast
 the difference in light intensity between the image and the adjacent background relative
to the overall background intensity.
 This can further be improved by staining the specimen. When the stains bind to the cells,
the contrast is increased
 This is achieved by use of lenses. There are two type of concave lenses used:
• Ocular lens with a magnification power of the 10X.
• Objective lens-
• scanning (4x), low power (10X),
• high power (40x) and oil immersion (100X).

 Bright-field OR Light Microscope
 Dark field Microscope
 Phase contrast Microscope
 Fluorescence Microscope
 Confocal Microscope
 Electron Microscope
 Inverted plane Microscope
PARTS OF MICROSCOPE TYPES OF MICROSCOPEPARTS OF MICROSCOPE TYPES OF MICROSCOPE

BRIGHT-FIELD
OR
LIGHT MICROSCOPE

The bright-field or light microscope forms a dark image against a
brighter background.
Working Principle
The rays emitted from the light source pass through the iris
diaphragm and fall on the specimen. The light rays passing through
the specimen is gathered by the objective and a magnified image is
formed. The image is further magnified by the ocular lens to produce
the final magnified virtual image.
BRIGHT-FIELD

BRIGHT-FIELD

Structure
The parts in a bright-field microscope are divided into three groups
Mechanical Parts.
 Base
 C-shaped arm
 Mechanical stage
Magnifying Parts
 Ocular lens
 Objective lens
Illuminating Parts
 Condenser:
 Iris diaphragm
 Light source
 Fine and coarse adjustment knobs
BRIGHT-FIELD

Applications
Bright-field microscope is used:-
• Viewing stained or naturally pigmented specimens
• Stained prepared slides of tissue sections or living photosynthetic organisms.
BRIGHT-FIELD

DARK-FIELD

DARK-FIELD
Dark field microscope, the object appears bright against a dark background.
 The dark field condenser has
 central opaque area that blocks Light from entering the objective lens directly.
 peripheral annular hollow area which allows the light to pass through and focus
on the specimen obliquely.

DARK-FIELD
Ray Diagram of Dark field

Applications
• Identify the living unstained cells and
• Thin bacteria like spirochetes, the most notorious bacterium
Trepnonema pallidum_the causative agent for syphilis, which cannot
be visualized by light microscopy.
DARK-FIELD
Trepnonema pallidum

Phase contrast
Microscope

 This microscope visualizes the unstained living cells by creating difference in
contrast between the cells and water.
 It converts slight differences in refractive index and cell density into easily
detectable variations in light intensity.
 The background, formed by un deviated light is bright, while the unstained object
appears dark and well defined.
Phase contrast

Phase contrast

Phase contrast
How phase contrast microscope works

Applications
Phase contrast microscopy is especially useful for studying:
 Microbial motility
 Determining the shape of living cells, and
 Detecting bacterial components, such as
 Endospores and
 Inclusion bodies which become clearly visible because they have refractive
indices markedly different from that of water.
Phase Contrast

Fluorescence
Microscope

The "fluorescence microscope" refers to any microscope that uses
fluorescence property to generate an image.
Principle of Fluorescence Microscope
Fluorescence Microscope

Applications
Epifluorescence microscope: it is the simplest format of
fluorescence microscope, which has the following
applications.
 Auto fluorescence: Certain microbes directly
fluoresce when placed wider uv lamp, e.g. Cyclospora
(a protozoanparasite).

• Microbes coated with fluorescent dye: Certain microbes fluoresce when they
are stained non-specifically by fluorochrome dyes.
• Acridine orange dye is used for the detection of malaria parasites by a method
called as quantitative buffy coat (QBC) examination.
• Auramine phenol is used for the detection of tuberclebacilli

Tubercle bacilli seen under fluorescence
microscope

Confocal Microscope
CONFOCAL
MICROSCOPE

Confocal Microscope
 Is an updated version of fluorescence microscopy.
 Confocal microscopy is an optical imaging technique for increasing optical resolution and
contrast of a micrograph.
 Uses pin hole screen to produce high resolution images.
 Eliminates out of focus
 So images have better contrast and are less hazy.
 a series of thin slices of the specimen are assemble is generate a three dimensional images.

Principle
In confocal microscopy two pin holes are typically used :-
• a pin hole is placed in front of the illumination source to allow transmission only through a
small area
• This elimination pin hole is imaged on to the focal plane of the specimen that is only a point
of a specimen is eliminated at one time
• Fluorescence excited in this manner at the focal plane is imaged on to a confocal pinhole
placed right in front of the detector.
• Only fluorescence excited within the focal plane of the specimen will go through the detector
pinhole
• Scanning of small sections is done and joined them together for better view .
Confocal Microscope

Confocal Microscope
Applications:
 Confocal microscopy allows analysis of fluorescent labelled thick specimen without
physical sectioning
 Three- Dimensional reconstruction of specimen.
 More color possibilities.
 Improved resolution.

Confocal Microscope

Electron
Microscope

An electron microscope (EM) uses accelerated electrons as a source
of illumination.
Because the wavelength of electrons can be up to 100,000 times
shorter than that of visible light photons.
Electron Microscope

• The EM has a much better resolving power than a light
microscope; hence, it can reveal the details of flagella, fimbriae
and intracellular structures of a cell.
• Electron microscopes are of two types:
1. Transmission electron microscope
2. Scanning electron microscope.
Electron Microscope

Transmission Electron Microscope
The TEM is somewhat analogous to the brightfield light microscope in
terms of the way it functions. However, it uses an electron beam from
above the specimen that is focused using a magnetic lens (rather than a
glass lens) and projected through the specimen onto a detector.
Working Principle

Applications
1. TEMs offer very powerful magnification and resolution.
2. TEMs have a wide-range of applications and can be utilized in a variety of
different and scientific, educational and industrial fields.
3. TEMs provides information on elements and compound structure.
4. Images are high-quality and detailed

 SEM uses electron beams to visualize surfaces; useful to observe the three-
dimensional surface details of specimens.
 Scanning electron microscopy is used for inspecting topographies of specimens at
very high magnifications.
 SEM magnifications can go to more than 300,000 X.
Scanning Electron Microscope (SEM)

principle

Applications
 Detailed 3D and topographical imaging and the versatile information garnered
from different detectors.
 Instrument works very fast.
 Modern SEM allow for the generation of data in digital form.
 Can magnify up to 100,000x

Scanning Tunneling
Microscope

Scanning tunnelling microscope
 A scanning tunneling microscope (STM) is an instrument for imaging
surfaces at the atomic level.
 The scanning tunneling microscope was invented in 1982 by Binnig and Rohrer.
 The instrument consists of a sharp conducting tip which is scanned across a flat
conducting sample.

Scanning tunnelling microscope
How STMWorks
 The STM uses a tip that ends in a single atom and a voltage is passed though the
tip and the sample.
 Electrons use a quantum mechanical effect to ‘tunnel’ from the tip to the
sample or vice versa.
 The current that results depends upon the distance between probe tip and
sample surface.
 The tip is attached to a piezoelectric tube and voltage applied to the piezo rod
is altered to maintain a constant distance for the tip from the surface.
 Changes in this voltage allows a three dimensional picture of the material
surface to be built up as the tip is scanned back and forth across the sample.

Scanning Tunnelling Microscope
Applications
 useful for studying friction,
 surface roughness, defects and surface reactions in materials like catalysts.
 STMs are also very important tools in research surrounding semiconductors and
microelectronics.

Inverted Plane
Microscope

Inverted Plane Microscopes

An inverted microscope is a microscope with its light
source and condenser on the top, above the stage pointing
down, while the objectives and turret are below the stage
pointing up.
It was invented in 1850 by J. Lawrence Smith, a faculty
member of Tulane University (then named the Medical
College of Louisiana).

Applications
 Inverted microscopy is a very popular technique for live cell
imaging. Here, living cells are observed through the bottom of a cell
culture vessel.
 Inverted microscope is also used for visualization of
the mycobacterium tuberculosis bacteria in the technique called
microscopic observation drug susceptibility assay (MODS).

MICROSCOPY BY INSHA JAN

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