Light Microscope : use sunlight or artificial
light.
1. Bright field microscope.
2. Dark field microscope.
3. Phase contrast microscope.
4. Fluorescence microscope.
Electron microscope : use of electron.
1. Transmission electron microscope.
2. Scanning electron microscope.
In light microscopy, light typically passes through a
specimen and then through a series of magnifying
lenses.
Microscopes are of great importance in the study of
microorganisms and biomolecules.
Light microscopes are simplest of all microscopes.
Light microscopes use lenses to bend and focus light
rays to produce enlarged images of small objects.
Bright-field Microscopy.
Dark-field Microscopy.
Phase contrast Microscopy.
Fluorescence Microscopy.
Light is transmitted and focussed by mirror and
condenser.
Focussed light illuminate the object or specimen.
The refracted light is collected by an objective
where primary image of the object is formed, it is
real,inverted enlarged image of the object.
The eyepiece further magnifies this primary
image into virtual,erect enlarged image, this is
the final image that lies above the stage.
Total magnification of specimen by multiplying the
objective lens magnification power by the ocular lens
magnification power.
Low power x 10, high power x 40 and oil immersion x
100
Is also called resolving power of image.
i.e the ability to distinguish that two objects are
separate and not one.
Resolving power of a microscope is determined by
the wave length of light entering the objective lens.
A general principle of ,microscopy is that the shorter
the wave length of light used in the instrument, the
greater the resolution
The white light used in a compound light
microscope has relatively long wave length and
cannot resolve structures smaller than about 0.2
µm.
Immersion oil is placed between the glass and
objective lens.
The immersion oil has the same refractive index
as glass of the microscope.
The oil enhances the resolution by preventing
light rays from dispersing and changing wave
length after passing through the specimens.
Observation of morphology of microorganisms.
Detection of cell structures.
Observation of intracellular structures.
Observation of motility.
Measurement of size.
Observation of blood smears.
The useful magnification of Light microscope is
limited by its resolving power.
The resolving power in limited by wavelength of
illuminating beam.
Resolution is determine by certain physical
parameters like wave length of light and light
generating power of the objective & condenser lens.
The ordinary microscope is called as a bright
field microscop.
It forms dark image against bright background.
Image is created by objective and ocular lenses
working together.
Light from illuminated specimen is focused by the
objective lens creating enlarged image within the
microscope.
The ocular lens further modifies the primary image.
Total magnification is calculated by magnification by
objective multiply by magnification by eyepiece.
Ex : 45x X 10x =450x
Bright field compound microscopes are commonly
used to view live and immobile specimens such as
bacteria, cells, and tissues. For transparent or
colorless specimens, however, it is important that
they be stained first so that they can be properly
viewed under this type of a microscope. Staining is
achieved with the use of a chemical dye. By applying
it, the specimen would be able to adapt the color of
the dye. Therefore, the light won’t simply pass
through the body of the specimen showing nothing
on the microscope’s view field
Dark field microscopy is frequently performed on the
same microscope on which bright-field microscopy is
performed.
Instead of the normal condenser that contains an
opaque disk.
The disk blocks light that would enter the objective
lens directly.
Only light that has been reflected or refracted by the
specimen forms the image
The field surrounding specimen appears dark while
the object brightly illuminated
The advantage of darkfield microscopy also becomes
its disadvantage: not only the specimen, but dust and
other particles scatter the light and are easily
observed
For example, not only the cheek cells but the bacteria
in saliva are evident.
The dark field microscopes divert illumination and
light rays thus, making the details of the specimen
appear luminous.
Dark field light microscopes provide good results,
especially through the examination of live blood
samples.
It can yield high magnifications of living bacteria and
low magnifications of the tissues and cells of certain
organisms.
Certain bacteria and fungi can be studied with the
use of dark field microscopes.
The principle of phase-contrast microscopy is based
on the wave nature of light rays and the fact that
light rays can be in phase or out of phase.
In a phase-constrast microscope, one set of light rays
comes directly from the light sources.
The other set comes from light that is reflected or
diffracted from a particular structure in the
specimen.
(diffraction is the scattering of light rays-direct rays
and reflected or diffracted rays are brought
together)
Both combined rays form an image of the specimen
on the ocular lens, containing areas that are relatively
light (in-phase), through shades of gray, to black(out
phase)
Through the use of annular lens, the differences in
phase are amplified so that in-phase light appears
brighter than out-of-phase.
To study unstained livingcells.
Detailed examination of internal structures in
living microorganism
To study flagellar movements and motility of
bacteria and protozoans.
To study intestinal and other living protozoa
such as amoeba and trichomonas.
To examine fungi grown in culture
Phase contrast microscopy is used in study of living
cells and tissues.
Microbes and parasites can be study .
Useful in observing cells cultured in vitro during
mitosis.
In all types of microscopes, cell constituents are not
distinguishable, although staining dose , but not
totally.
In fluorescent microscopy, various fluorescent dyes
are used which gives property of fluorescence to only
specific part of the cell and hence it can be focused.
When certain compounds are illuminated with high
energy light, they then emit light of a different, lower
frequency. This effect is known as fluorescence.
Often specimens show their own characteristic
autofluorescence image, based on their chemical
makeup.
Many different fluorescent dyes can be used to
stain different structures or chemical
compounds.
One particularly powerful method is the
combination of antibodies coupled to a
fluorochrome as in immunostaining.
Examples of commonly used fluorochromes are
fluorescein or rhodamine
A component of interest in the specimen is
specifically labeled with a fluorescent molecule
called a fluorophore
The specimen is illuminated with light of a specific
wavelength (or wavelengths) which is absorbed by
the fluorophores, causing them to emit longer
wavelengths of light (of a different color than the
absorbed light).
Typical components of a fluorescence microscope
are the light source (xenon arc lamp or mercury-
vapor lamp), the excitation filter, the dichroic
mirror and the emission filter.
The illumination light is separated from the
much weaker emitted fluorescence through the
use of an emission filter.
The filters and the dichroic are chosen to match
the spectral excitation and emission
characteristics of the fluorophore used to label
the specimen.
In this manner, a single fluorophore (color) is
imaged at a time. Multi-color images of several
fluorophores must be composed by combining
several single-color images.
Fluorescence microscopy is a critical tool for
academic and pharmaceutical research, pathology,
and clinical medicine.
Electron microscopy is in some ways
comparable to light microscopy. Rather
than using glass lenses, visible light, and
the eye to observe the specimen, the
electron microscope uses electromagnetic
lenses, electrons, and a fluorescent screen
to produce the magnified image. That
image can be captured on photographic
film to create an electron photomicrograph.
The superior resolution of the electron
microscope is due to the fact that electrons have
a much shorter wavelenght than the photons of
white light.
The electron beam is focused by circular electron
magnets, which are analogous to the lenses in
the light microscope. The object which is held in
the path of the beam scatters the electrons and
produces an image which is focused on a
fluorescent viewing screen.
A. Transmission Electron Microscope
B. Scanning Electron Microscope
A. Transmission Electron Microscope
In a transmission microscope, electrons pass through
the specimen and scattered. Magnetic lenses focus
the image onto a fluorescent screen or photographic
plate.
Electron light pass directly through the specimen
that has been prepared by thin sectioning, freeze
fracturing, or freeze etching.
It is used to observe fine details of cell structure.
B. Scanning Electron Microscope
In a scanning electron microscope, primary
electron sweep across the specimen and knock
electrons from its surface.
The second electron is picked up by a collector,
amplified, and transmitted onto viewing screen
or photographic plate.
It scan a beam of electrons back and forth over
the surface of a specimen producing three-
dimensional views of the surfaces of whole
microorganism.
Spirillum volutans. Electron
micrograph showing individual
flagella
Leptospira biflexa. Electron
micrograph showing axial filament .
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