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Contents
Introduction
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2
Aristotle
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3
Compound microscope
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3
20th century
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Electron microscope
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Bright field microscopy
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Performance
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Limitations
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FLUORESCENCE MICROSCOPY:
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Light sources:
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The fluorescent antibody technique_ Immunofluorescence
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Fluorescent proteins:
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PHASE CONTRAST MICROSCOPY
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Applications:
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Phase-contrast microscopy
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Applications:
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Transmission Electron Microscopy
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Advantages:
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Scanning Electron Microscopy
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Disadvantages of SEM
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Advantages and disadvantages
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References
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Microscopy is the technical field of using microscopes for viewing the objects and areas of objects that
cannot be observed or viewed with naked eye
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There are three well known branches of microbiology namely:
Optical microscopy
Scanning probe microscopy
X ray microscopy

Brief History
The origin of the word microscope according to the Online Etymology Dictionary is as follows: 1656,
from Mod
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microscopium, lit
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micro- (q
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) + -

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skopion
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" Microscopic "of minute size" is attested from
1760s
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Aristotle
In history the first probable person to desribe the working of microscope is Aristotle
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Greeks, however,used these lenses for surgical purposes to cauterize the wounds and
lesions caused by leprosy and so forth
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The year is not verified
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In
the late 1590s, they used several lenses in a tube and were amazed to see that the object at the end of
the tube was magnified significantly beyond the capability of a magnifying glass
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That is to say, they had discovered that an image magnified by a single lens
can be further magnified by a second or more lenses
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Hooke was a sickly genius who loved to
experiment
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He
invented the universal joint, the iris diaphragm (another key component of many modern light
microscopes), a respirator, an anchor escapement and balance spring for clocks
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He discovered that by using a second lens of
different shape and refracting properties, he could realign colors with minimal impact on the
magnification of the first lens
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Combined,
these two discoveries contributed towards a marked improvement in the quality of image
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It is worth remembering that up until now, each new stride has been in the quality or application of the
lenses
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20th century
At the turn of the 19th/20th centuries Louis Pasteur invented pasteurization while Robert

Koch discovered his famous or infamous postulates: the anthrax bacillus, the tuberculosis bacillus and
the cholera vibrio
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In 1904, Zeiss overcame this limitation with the introduction the first commercial UV
microscope with resolution twice that of a visible light microscope
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Spurned by Zeiss, his phase contrast
innovation was not introduced until 1941 although he went on to win a Nobel Prize for his work in 1953
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Physics dictates that light microscopes are limited by the physics of light
to 500x or 1000x magnification and a resolution of 0
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Techniques of microscopy
Huge techniques of microscopy are used to improve the quality of observation of specimen
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Sample illumination is via
transmitted white light, i
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illuminated from below and observed from above
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The simplicity of the technique and the minimal sample preparation required are significant
advantages
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The light path therefore consists of:
a transillumination light source, commonly a halogen lamp in the microscope stand;
a condenser lens, which focuses light from the light source onto the sample;
an objective lens, which collects light from the sample and magnifies the image;
oculars and/or a camera to view the sample image
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Performance
Bright-field microscopy typically has low contrast with most biological samples, as few absorb light to a
great extent
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Bright-field illumination is useful for samples that have an intrinsic color, for
example chloroplasts in plant cells
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Living cells can be seen with bright-field microscopes
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The practical limit to magnification with a light microscope is around 1300X
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[2]
Low apparent optical resolution due to the blur of out-of-focus material
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g
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These samples often have to be stained before viewing
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g
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Advancements
Reducing or increasing the amount of the light source by the iris diaphragm
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Immersion oil has the same refraction as glass and improves the resolution of the observed
specimen
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Use of a colored (usually blue) or polarizing filter on the light source to highlight features not visible
under white light
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FLUORESCENCE MICROSCOPY:
The term fluorescence was coined by sir George Gabriel stokes (1819-1903)
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Stokes used the light from the sun as the source of
excitation
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Such substances are called fluorescent and the phenomenon is termed fluorescence
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The light that is responsible for excitation, or
moving the electron to a higher energy state, is of shorter wavelength and higher energy than the
fluorescence emission, which has a longer wavelength, lower energy, and different color
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With fluorescence microscopy, scientists can observe the location of specific cell types
within tissues or molecules within cells
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Exciter filters transmits
only blue light to the specimen and blocks out all other colors
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Specimen is
stained with a fluorescent dye: few portions of specimen retain the dye, others do not
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It allows green light to pass to the eye, however, it blocks out any residual
blue light from the specimen which may not have been completely deflected by the dichroic mirror
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0)
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Barrier filters are selected on the basis of dye used
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0_ special features of fluorescence microscopy
Light sources:
It requires intense, near- monochromatic, illumination which widespread light sources, like halogen
lamps cannot provide
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Sample preparation
In order for a sample to be suitable for fluorescence microscopy it must be fluorescent
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e
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Antibodies to which a fluorescent dye is attached termed as labeled antibodies
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Bacterial cells that have combined with labeled antibody will be visible in the microscopic
preparation
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A fluorophore can be directly conjugated to the primary antibody
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For example, a
primary antibody raised in a mouse which recognizes tubulin combined with a secondary anti-mouse
antibody derivatised with a fluorophore could be used to label microtubules in a cell
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Some of these are small molecules which
are intrinsically fluorescent and bind to biological molecule of interest
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There are many fluorescent molecules called fluorophore or fluorochromes such as fluorescein, alexa
fluors which linked to a different molecule which binds the target of interest within the sample
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In biological sample, it directly makes a protein of
interest fluorescent
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Limitations:
Photo bleaching: fluorophores lose their ability to fluoresce as they are illuminated in a process called
photo bleaching
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It can be reduce by using photo protective scavenger chemicals or by minimizing
illumination
Unlike transmitted and reflected light microscopy techniques fluorescence microscopy only allows
observation of specific structures which have been labeled for fluorescence
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In early 1930s, it was invented by Frits Zernike
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Principle:
This technique is based on the fact that light passing through one material and into another material of
a slightly different refractive index and thickness will undergo a change in phase
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Applications:
This technique is extremely valuable for studying living unstained cells and widely used in applied
biology
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This special optical system makes it possible to distinguish unstained structures within a cell
which differ only a slightly in their refractive indices or thickness
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In 1952 Georges patented what is today known as differential interference contrast
(DIC) microscopy
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But DIC microscopy and phase contrast microscopy both use polarized light, which is unsuitable
when the object or its container alter polarization
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Traditional phase-contrast methods enhance contrast optically, blending brightness and phase
information in single image
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These methods digitally create two separate images, an ordinary bright-field image and a
so-called phase-shift image
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Phase-contrast microscopy
First described in 1934 by Dutch physicist Frits Zernike,is an optical microscopy technique that
converts phase shifts in light passing through a transparent specimen to brightness changes in the
image
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Principle
Unstained living cells absorb practically no light
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This makes the cells barely, or not at all, visible in a
bright field microscope
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In a phase contrast microscope, these phase shifts are converted into changes in
amplitude, which can be observed as differences in image contrast
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Cilia and
flagella, for example, are nearly invisible in bright field but show up in sharp contrast in phase contrast
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Most living
microscopic organisms are much more obvious in phase contrast
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Limitations Phase contrast condensers and objective lenses add considerable cost to a microscope,
and so phase contrast is often not used in teaching labs except perhaps in classes in the health
professions and in some university undergraduate programs
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To use phase contrast the light path must be aligned
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This usually involves sliding a component into the light path
or rotating a condenser turret
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Generally, more light is needed for phase contrast than for
corresponding bright field viewing, since the technique is based on a diminishment of brightness of most
objects
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Roughly a decade later, the first electron microscope picture of eukaryotic cells was taken by Keith
Porter
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Working
TEMs employ a high voltage electron beam in order to create an image
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Rather than having a glass lens
focusing the light (as in the case of light microscopes), the TEM employs an electromagnetic lens which
focuses the electrons into a very fine beam
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An
image of the specimen with its assorted parts shown in different shades according to its density appears
on the screen
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Fig
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Advantages:
The advantage of the transmission electron microscope is that it magnifies specimens to a much higher
degree than an optical microscope
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For biologists, the interior workings of cells, such as
mitochondria and organelles, are clearly visible
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Disadvantages:
The transmission electron microscope requires that specimens be put inside a vacuum chamber
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Some delicate samples may also be damaged by the electron beam and must first be stained
or coated with a chemical to protect them
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Scanning Electron Microscopy
The scanning electron microscope (SEM) uses a focused beam of high-energy electrons to generate a
variety of signals at the surface of solid specimens
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In most
applications, data are collected over a selected area of the surface of the sample, and a 2-dimensional
image is generated that displays spatial variations in these properties
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Advantages of SEM:
The advantages of a scanning electron microscope include its wide-array of applications, the
detailed three-dimensional and topographical imaging and the versatile information garnered
from different detectors
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This instrument
works fast, often completing SEI, BSE and EDS analyses in less than five minutes
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Although all samples must be prepared before placed in the vacuum chamber, most SEM
samples require minimal preparation actions
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SEMs are expensive, large and must
be housed in an area free of any possible electric, magnetic or vibration interference
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The preparation of samples
can result in artifacts
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There is
no absolute way to eliminate or identify all potential artifacts
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Finally, scanning electron microscopes
carry a small risk of radiation exposure associatedwith the electrons that scatter from beneath the
sample surface

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