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.
5. Transmission electron microscope.
6. Scanning electron microscope.
In light microscopy, light typically passes through a
specimen and then through a series of magnifying
lenses.
Microscopesare of great importance in the study of
microorganisms and biomolecules.
Light microscopes are simplest of all microscopes.
Lightmicroscopes 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.
[Link] 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 generalprinciple 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.
Theoil enhances the resolution by preventing
light rays from dispersing and changing
wave length after passing through the
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.
Resolutionis 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.
Imageis created by objective and ocular lenses
working together.
Lightfrom illuminated specimen is focused by the
objective lens creating enlarged image within the
microscope.
Theocular lens further modifies the primary
image.
Totalmagnification 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
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.
Oftenspecimens 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.
SAMPLE PREPARATION
In order for a sample to be suitable for fluorescence microscopy it must be
fluorescent. There are several methods of creating a fluorescent sample; the
main techniques are labelling with fluorescent stains or, in the case of biological
samples, expression of a fluorescent protein.
Biological fluorescent stains
Many fluorescent stains have been designed for a range of biological molecules.
Some of these are small molecules which are intrinsically fluorescent and bind a
biological molecule of interest.
Major examples of these are nucleic acid stains such as DAPI and Hoechst
(excited by UV wavelength light) and DRAQ5 and DRAQ7 (optimally excited by
red light) which all bind the minor groove of DNA, thus labeling the nuclei of
cells.
Others are drugs, toxins, or peptides which bind specific cellular structures and
have been derivatied with a fluorescent reporter.
There are many fluorescent molecules called fluorophores or fluorochromes such as
fluorescein, Alexa Fluors, or DyLight 488, which can be chemically linked to a different
molecule which binds the target of interest within the sample.
Immunofluorescence is a technique which uses the highly specific binding of an
antibody to its antigen in order to label specific proteins or other molecules within
the cell. A sample is treated with a primary antibody specific for the molecule of
interest. A fluorophore can be directly conjugated to the primary antibody.
Fluorescent proteins
The modern understanding of genetics and the techniques available for modifying
DNA allow scientists to genetically modify proteins to also carry a fluorescent
protein reporter. In biological samples this allows a scientist to directly make a
protein of interest fluorescent. The protein location can then be directly tracked,
including in live cells.
Phase contrast Microscopy
[Link]
THANK
