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Microscopy
Microscopy is the technical field of using microscope to view objects and areas
of objects that cannot be seen with the naked eye (objects that are not within
the resolution range of the normal eye) ,the word "microscopy" comes from
Greek roots: mikros, small + skopeo, to view = to view small (objects)
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Since its invention, the microscope has been a
valuable tool in the development of scientific theory
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A
compound microscope is composed of two elements; a primary magnifying lens
and a secondary lens system, similar to a telescope
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The microscope is used in many forensic disciplines including firearms
identification, serological examinations, drug chemistry, and trace evidence
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The trace evidence analyst may encounter limitless types
of particulate matter including fibers, hairs, paint, explosives, pollen, soil,
glass,and tape
The English scientist, Robert Hooke (1635-1703), was the first to publish work
based upon the use of an optical microscope

Diagram of a simple microscope

BASIC PRINCIPLES OF MICROSCOPY

1)Resolution

The magnification of small things is a necessary facet of forensic research, but
the fine detail in components requires that any imaging system be capable of
providing spatial information across small distances
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Resolution is best when the distance separating the two tiny objects is
small
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A simple mathematical equation defines the smallest distance (dmin) separating
the two very small objects:
dmin = 1
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A
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A
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A
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2)Magnification

Magnification is the process of enlarging something only in appearance, not in
physical size
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Most microscopes in
current use are known as compound microscopes, where a magnified image of
an object is produced by the objective lens, and this image is magnified by a
second lens system (the ocular or eyepiece) for viewing
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Objective magnification
powers range from 4X to 100X
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For instance, using a 5X objective with a 10X eyepiece yields a total
visual magnification of 50X and likewise, at the top end of the scale, using a
100X objective with a 30X eyepiece gives a visual magnification of 3000X
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It is not only
necessary to obtain bright light around the object, but for optimal imaging, the
light should be uniform across the field of view
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The front element of the condenser is usually a large, flattened lens that sits
directly beneath the specimen
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Two apertures in the
illumination system allow you to regulate the diameter of the illumination
beam by closing or opening iris diaphragms
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The
second of these diaphragms, known as the field aperture diaphragm, does not
affect resolution as dramatically and is regularly adjusted for optimal
illumination
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Operationally, it is easy to obtain optimal illumination by first
placing any specimen on the stage and focusing on the object
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Next, raise or lower the
condenser until the edges of the field aperture diaphragm are clearly focused
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It may be necessary to center the field aperture diaphragm, using
the condenser centering screws
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DESCRIPTION AND FORENSIC UTILITY OF DIFFERENT MICROSCOPES
1) Simple microscope

A microscope is a scientific instrument which only has one lens, as opposed to
the compound lenses used in more complex microscope designs that makes
things normally too small to see look bigger, so they can be seen better and
examined correctly
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The earliest microscopes had only 1 lens and
are called simple microscopes
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This design is classically used for
basic microscopes used to introduce children to science and microscopy, and
they may be utilized in some industries as well
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Modern
simple microscopes are usually hand held, designed for field work or quick
viewing of objects which require magnification
Forensic utility :
Simple magnifying glass can be used at crime scenes to get an enhanced view
of the fingerprints
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compound microscope is a microscope which uses a lens close to the object
being viewed to collect light (called the objective lens) which focuses a real
image of the object inside the microscope That image is then magnified by a
second lens or group of lenses (called the eyepiece) that gives the viewer an
enlarged inverted virtual image of the object
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Forensic utility:
Blood analysis is undertaken, which is of immense help in diagnosing illnesses
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Various crime cases are detected and solved by drawing out human cells and
examining them under the microscope in forensic laboratories
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Forensic experts and scientists can also find out the country from which a
particular drug has come by viewing its particles under a compound
microscope
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It
consists of two microscopes connected by an optical bridge, which results in a
split view window enabling two separate objects to be viewed simultaneously
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Although the human eye can be very good at discerning minute differences in
color and morphology, the brain has a more difficult time remembering and
processing these subtle differences
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Reflected light instruments are used by firearms
examiners to compare rifling marks on bullets as well as ejector marks and
firing pin impressions on cartridge cases
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Transmitted light microscopes are used to compare hairs, fibers and layered
paint chips which have been thin-sectioned
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Human hair comparisons, particularly
the final stages of an examination, are conducted almost exclusively under a
comparison microscope
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Gravelle, a chemist, developed a comparison
microscope for use in the identification of fired bullets and cartridge cases with
the support and guidance of forensic ballistics pioneer Calvin Goddard
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The firearm from which a bullet or cartridge case has been fired is
identified by the comparison of the unique striae left on the bullet or cartridge
case from the worn, machined metal of the barrel, breach block, extractor, or
firing pin in the gun
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"As long as
he could inspect only one bullet at a time with his microscope, and had to keep
the picture of it in his memory until he placed the comparison bullet under the
microscope, scientific precision could not be attained
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" Calvin Goddard
perfected the comparison microscope and subsequently popularized its use
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Phase shifts themselves are invisible, but become visible
when shown as brightness variations
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Changes in amplitude (brightness)
arise from the scattering and absorption of light, which is often wavelength
dependent and may give rise to colors
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Without special arrangements,
phase changes are therefore invisible
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Unstained
biological specimens, such as living cells, are essentially transparent to our
eyes, but they interact with light in a fairly uniform way, by retarding (slowing)
the passage of a light beam by approximately 1/4 of a wavelength ( )
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Intensity (amplitude) is additive and light rays that are 1/2 out of
phase are perceived as darkness
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The phase contrast microscope was invented by Zernicke in
the 1930's as a means to generate contrast in biological specimens, changing
these invisible phase differences into visible amplitude differences
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To separate the
beams of light from each other, he placed a transparent ring (known as an
annulus) in an opaque disk and inserted this disk into the optical path of the
microscope, within the condenser
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Nearly all of the light that passes through the sample but misses
the specimen then passes through the objective lens through this ring
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He designed the glass
plate holding the ring so that all light missing the ring would encounter an
additional 1/4
of retardation relative to the beams of light that had not
interacted with the specimen, placing the light rays that had interacted with
the specimen out of phase with rays that had not interacted with the specimen
by 1/2
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Forensic utility
The phase contrast microscope is used primarily in serological and glass
examinations by forensic experts
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it
can also be used in Forensic Detection of Sperm from Sexual Assault Evidence
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The instrument uses two separate optical paths with two objectives and
eyepieces to provide slightly different viewing angles to the left and right eyes
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The stereo microscope is often used to study the surfaces of solid specimens or
to carry out close work such as dissection, microsurgery, identification of
circuit board and forensic engineering
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Stereo

microscopes are essential tools in entomology
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Use of reflected light from the object allows examination of specimens
that would be too thick or otherwise opaque for compound microscopy
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Modern stereomicroscope optical design
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Documents, another area of
forensics, are also inspected through a macroscope or stereomicroscope –
though not necessarily a comparison instrument
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Detail of a bank note (embossed elements)
document: pen over toner
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Although originally used
predominantly in the field of geology, it has recently become more widely used
in medical and biological research fields too
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It uses polarising filters to make use of polarised light, configuring
the movement of light waves and forcing their vibration in a single direction
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This is the type of light
that we see, and its waves vibrate in random directions
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This type of light is used in polarising microscopy to improve image quality
when examining birefringent (doubly-refracting), anisotropic materials
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One such example is wood, which breaks more
easily in a direction along its grain than against it
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How does this translate to optical terms? In general, most liquids and gases are
isotropic and have the same optical properties in all directions: i
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, they have
one refractive index
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Their
optical properties vary depending on the orientation of incident light (light that
falls on a surface), and they have numerous refractive indices
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e
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Generally, you will need
two additional for polarising microscopy:



Polariser: This filter can be manually rotated and is placed somewhere
in the light path beneath the specimen, usually below the stage
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Consequently, it only allows



light waves vibrating along their polarising axis to completely pass
through, while absorbing light waves that move in other directions
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Most student
microscopes have an analyser that is fixed in place, usually mounted
above the stage
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Depending on the microscope, however, you may
be able to rotate the polarise, or push it in or out of the light path,
according to your needs
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So when you turn on the

microscope’s light source, light moves upwards and is polarised to move in one
direction by the polariser
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Light waves then project through the specimen, and vibrate in an east-west
position
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When the analyser is pushed in, only light moving in a north-south position
can to move through
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Because all of the light was already polarised to move in an eastwest position only, no light can pass
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Uses and forensic utility of Polarised Light Microscopy
You can use this technique to highlight the features of various substances
such as crystals, fibers, and minerals, which can aid in their identification
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When adjusted, however, light is able to pass through the filters at
different angles, allowing you to see different aspects of the specimen
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Not only does it
perform all the duties of a normal brightfield microscope for the study of
morphology, but it also permits observations and measurements in plane
polarized light and between crossed polars
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Since the components of a birefringent, anisotropic sample are aligned at
different angles, rotating the polariser (or the rotating stage, if your microscope
has one) will cause different parts to ―black out‖ at different times
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The "fluorescence
microscope" refers to any microscope that uses fluorescence to generate an
image, whether it is a more simple set up like an epifluorescence microscope,
or a more complicated design such as a confocal microscope, which uses
optical sectioning to get better resolution of the fluorescent image
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e
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The illumination
light is separated from the much weaker emitted fluorescence through the use
of a spectral emission filter
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The filters and the dichroic are chosen to match the spectral excitation
and emission characteristics of the fluorophore used to label the specimen
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Multi-color images of several types of fluorophores must be composed by
combining several single-color images
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First, the microscope has a filter that only lets through radiation with
the desired wavelength that matches your fluorescing material
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When they relax to a lower level, they emit light
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Here, the
fact that the emitted light is of lower energy and has a longer wavelength is
used
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Light sources
Fluorescence microscopy requires intense, near-monochromatic, illumination
which some widespread light sources, like halogen lamps cannot provide
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Lasers are most widely used for more complex fluorescence
microscopy
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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
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e
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[1] In the
life sciences fluorescence microscopy is a powerful tool which allows the
specific and sensitive staining of a specimen in order to detect the distribution
of proteins or other molecules of interest
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DNA is stained blue, a protein called INCENP is green, and the microtubules
are red
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Forensic utility
Fluorescence microscopy is of much consideration and importance in field of
forensic biotechnology where the application of fluorescence microscopy in
microbial cell biology has advanced the field dramatically
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The quantitative
nature and high temporal resolution of fluorescence microscopy make it
particularly useful for studies of intracellular dynamic systems, such as
signaling networks
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Several techniques allow researchers to
follow real-time dynamics of protein interactions, at steady state or upon
stimulation, and therefore to investigate signal propagation, amplification, and
integration in the cell
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8) Scanning Electron microscope
The scanning electron microscope (SEM) is a type of electron microscope that
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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The
SEM is also capable of performing analyses of selected point locations on the
sample; this approach is especially useful in qualitatively or semi-quantitatively
determining chemical compositions, crystalline structure, and crystal
orientations
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The number of secondary electrons
depends on the angle at which beam meets surface of specimen, i
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on
specimen topography
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Principles and capacities
Accelerated electrons in an SEM carry significant amounts of kinetic energy,
and this energy is dissipated as a variety of signals produced by electronsample interactions when the incident electrons are decelerated in the solid
sample
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Secondary electrons
and backscattered electrons are commonly used for imaging samples:
secondary electrons are most valuable for showing morphology and topography
on samples and backscattered electrons are most valuable for illustrating
contrasts in composition in multiphase samples (i
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for rapid phase
discrimination)
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As the excited electrons return to lower energy states, they yield X-rays
that are of a fixed wavelength (that is related to the difference in energy levels
of electrons in different shells for a given element)
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SEM analysis is considered to be "non-destructive"; that is, x-rays
generated by electron interactions do not lead to volume loss of the sample, so
it is possible to analyze the same materials repeatedly
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In the most common or standard detection mode, secondary electron
imaging or SEI, the SEM can produce very high-resolution images of a sample
surface, revealing details less than 1 nm in size
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This is exemplified by the micrograph of pollen
shown above
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Back-scattered electrons (BSE) are beam electrons that are reflected from the
sample by elastic scattering
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BSE
images can provide information about the distribution of different elements in
the sample
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Characteristic X-rays are emitted when the electron beam removes an inner
shell electron from the sample, causing a higher-energy electron to fill the shell
and release energy
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Sample preparation
All samples must be of an appropriate size to fit in the specimen chamber and
are generally mounted rigidly on a specimen holder called a specimen stub
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For conventional imaging in the SEM, specimens must be electrically
conductive, at least at the surface, and electrically grounded to prevent the
accumulation of electrostatic charge at the surface
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Nonconductive specimens tend to charge when scanned by the electron
beam, and especially in secondary electron imaging mode, this causes
scanning faults and other image artifacts
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Conductive materials in current use for specimen coating include gold,
gold/palladium alloy, platinum, osmium, iridium, tungsten, chromium
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Unlike optical and transmission
electron microscopes, image magnification in the SEM is not a function of the
power of the objective lens
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Provided the electron gun can generate a beam with sufficiently
small diameter, a SEM could in principle work entirely without condenser or
objective lenses, although it might not be very versatile or achieve very high
resolution
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Forensic utility
Scanning Electron Microscopes in Forensic Investigations
The field of forensic investigation is of increasing importance and thus the role
of the Scanning Electron Microscope (SEM) becomes progressively more
significant
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Together with the ability to analyse the elemental composition of even the
smallest features on specimens, it becomes possible to make conclusive
identifications of the origin of some materials and thus contribute to the chain
of evidence
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An image is formed from the
interaction of the electrons transmitted through the specimen; the image is
magnified and focused onto an imaging device, such as a fluorescent screen, on
a layer of photographic film, or to be detected by a sensor such as a CCD
camera
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This
enables the instrument's user to examine fine detail—even as small as a single
column of atoms, which is thousands of times smaller than the smallest
resolvable object in a light microscope
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TEMs find
application in cancer research, virology, materials science as well as pollution,
nanotechnology, and semiconductor research
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At higher magnifications complex
wave interactions modulate the intensity of the image, requiring expert analysis
of observed images
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The first TEM was built by Max Knoll and Ernst Ruska in 1931, with this
group developing the first TEM with resolution greater than that of light in
1933 and the first commercial TEM in 1939
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What you
can see with a light microscope is limited by the wavelength of light
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You
can see objects to the order of a few angstrom (10-10 m)
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The possibility for high magnifications has made the TEM a valuable tool in
both medical, biological and materials research
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Instead of glass lenses
focusing the light in the light microscope, the TEM uses electromagnetic lenses
to focus the electrons into a very thin beam
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Depending on the density of the
material present, some of the electrons are scattered and disappear from the
beam
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The
image can be studied directly by the operator or photographed with a camera
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TEMs produce high-resolution, two-dimensional images,
allowing for a wide range of educational, science and industry applications
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Air needs to be pumped out of the vacuum chamber, creating a space where
electrons are able to move
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These solenoids are tubes with coil wrapped around
them
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The image can be manipulated by adjusting the voltage of the gun to
accelerate or decrease the speed of electrons as well as changing the
electromagnetic wavelength via the solenoids
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During transmission, the speed of electrons
directly correlates to electron wavelength; the faster electrons move, the shorter
wavelength and the greater the quality and detail of the image
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These differences provide information on the structure,
texture, shape and size of the sample
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They need to be sliced thin enough for electrons to
pass through, a property known as electron transparency
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Types of preparation include dehydration, sputter coating of
non-conductive materials, cryofixation, sectioning and staining
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TEMs provide topographical, morphological, compositional and

crystalline information
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This
information is useful in the study of crystals and metals, but also has
industrial applications
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Technology
companies use TEMs to identify flaws, fractures and damages to micro-sized
objects; this data can help fix problems and/or help to make a more durable,
efficient product
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Although most forensic scientists use microscopes at one time or another, the
forensic microscopist uses microscopes to locate, recover, identify and compare
trace evidence on a daily basis
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TEM can be
very useful in forensic trace evidence analysis due to its ability to analyze the
morphology of small particles, gather elemental information on very small
particles and determine the internal structure of small particles



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