The Microscope
• Compound microscope.
• Magnification, field of view, working distance,
and depth of focus.
• Comparison microscope.
• Advantages of stereoscopic microscope.
• Plane-polarized light and polarizing microscope.
• Advantages of linking a microscope to a
spectrophotometer.
• Utilizing a microspectrophotometer for
examining trace physical evidence.
• Mechanism of image formation for light
microscope Vs scanning electron
microscope (SEM).
• Advantages and applications of SEM in
forensic science.
• Virtual image: an image cannot be seen
directly. It can only be seen by a viewer
looking through a lens.
• Transmitted illumination: light that passes
up from the condenser and though the
specimen
• Real image: an image formed by the actual
convergence of light rays upon a screen
• Objective lens: the lower lens of a
microscope that is positioned directly over
the specimen
• Eyepiece lens: the lens of a microscope
into which the viewer looks; same as the
ocular lens
• Vertical or reflected illumination:
illumination of a specimen from above; in
microscopy it is used to examine opaque
specimens
• Condenser: lens system located under the
microscope stage that focuses light onto the
specimen
• Parfocal: construction of a microscope
such that when an image is focused with
one objective in position, the other objective
can be rotated into place and the field will
remain in focus
• Monocular: a microscope with one
eyepiece
• Binocular: a microscope with two
eyepieces
• Field of view: the area of the specimen
that can be seen after it is magnified
• Depth of Focus: the thickness of a
specimen entirely in focus under a
microscope
• Plane-Polarized light: light confined to a
single place of vibration
• Polarizer: a device that permits the
passage of light waves vibrating in only one
plane
• Microspectrophotometer: an instrument
that links a microscope to a spectrometer
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Compound Microscope
Polarizing Microscope
Comparison Microscope
Stereoscopic Microscope
Cell division in a frog's egg.
Microspectrophotometer
Scanning Electron Microscope (SEM)
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SEM Data
The Microscope
• Provides a direct image of a small object of
interest
– spectroscopy gives an abstract representation
which must be interpreted on the basis of a
model or some assumptions
• A typical animal cell is 10-20 nm in
diameter
Nanoscaled polyimide
structures
Side-wall morphology of solar cell
gridline
The Microscope
• Produce a magnified
image of a specimen
• Separate the details in
the image
• Render the details
visible to the human
eye or camera
– 5x smaller than the smallest object that can be
seen directly by the naked eye
Lenses
Refraction of a light ray as it passes through a prism
Lenses
Focal Point & Focal Length
• Light passing through two “identical” prisms stacked base
to base would intersect at point I
• The point at which parallel rays are converged to an image
is the focal point of the lens
• The distance of this point from the lens is the focal length
– produce a real image
– converging lens
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Simple Magnifier
The Compound Microscope
• Object O is placed
close to the lens
– rays converge but do
not intersect
– real image not formed
• The observer’s eye
follows rays back to
the point of apparent
origin (I)
• I bigger than object
• Rays pass first through the objective lens forming
a real, slightly enlarged, inverted image
• The second lens (eyepiece) acts as a simple
magnifier
Compound Microscope
• Both lenses produce
magnification
• Overall magnification
is found by
multiplying the two
magnifications
• Magnification
determined mainly by
objective
The Comparison Microscope
• Two compound microscopes combined into
one unit
• When viewer looks through the eyepiece, a
field divided into two equal parts is
observed
The Comparison Microscope
• Bullet comparisons
• Hair & Fiber comparisons
• Questioned documents
– specimen on left scope on left side of field
– specimen on right scope on right side of field
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Test Fire Reference Gun
Use A Comparison Microscope
Striations match
Stereoscopic Microscope
Stereoscopic Microscope
• Two separate
monocular
microscopes
• Each has its own set of
lenses
Using the Stereo Microscope
Using the Compound
Microscope
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FT-IR Microspectrophotometer
Photocopier Toner Analysis
• important for establishing corroborative evidence
linking documents to specific locations in forensic
investigations of corporate crime
• Must be performed non-destructively
– can’t remove toner from paper
– physical size of specimen is very small
• microscope to find sample
• FT-IR to analyze the sample
Photocopier Toner Analysis
Limitations of Light Microscope
• Radiation of a given wavelength can’t be
used to probe structural details much
smaller than its own wavelength
• Light Microscope
– limited to range of visible light
• 0.4 mm (violet) to 0.7 mm (deep red)
– bacteria & nitochondria (~0.5mm wide) smallest objects
that can be seen clearly
Range of Readily Resolvable
Objects
Scanning Electron Microscope
• This scanning electron microscope has a magnification
range from 15x to 200,000x and a resolution of 5
nanometers
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How the SEM Works
Conventional light microscopes use a series of glass lenses
to bend light waves and create a magnified image.
• The Scanning Electron
Microscope creates the
magnified images by
using electrons instead
of light waves
The SEM shows very detailed 3-dimensional images at
much higher magnifications than is possible with a light
microscope. The images created without light waves are
rendered black and white
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Samples have to be prepared carefully to withstand the
vacuum inside the microscope
• Biological specimens
are dried in a special
manner that prevents
them from shriveling.
• Because the SEM
illuminates them with
electrons, they also
have to be made to
conduct electricity
• How do you make a
mosquito conductive?
• SEM samples are
coated with a very thin
layer of gold by a
machine called a
sputter coater
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The specimen is now prepared
• Air is pumped out of the
column
• An electron gun [at the
top] emits a beam of high
energy electrons.
– travels downward through a
series of magnetic lenses
designed to focus the
electrons to a very fine spot
• As the electron beam
hits each spot on the
sample, secondary
electrons are knocked
loose from its surface.
• A detector counts
these electrons and
sends the signals to an
amplifier
The sample is placed inside the microscope's vacuum
column through an air-tight door
• Near the bottom, a set
of scanning coils
moves the focused
beam back and forth
across the specimen,
row by row
• The final image is
built up from the
number of electrons
emitted from each spot
on the sample
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Fiber Analysis
Energy-Dispersive X-Ray
Analysis (EDX)
• Electron beam ~5,00020,000eV
• Atomic electrons are
dislodged ionizing the
sample
• Resulting electron vacancy
filled by an electron from a
g=higher shell
• X-ray is produced
– x-ray energy
characteristic of the
parent atom
Gunshot Residue by EDX
• Residue particle from the hand of a person who fired a
.380 Browning automatic
• The peaks of lead, barium & antimony together with the
shape of the particle are quite specific & show that the
subject had fired a weapon
Who am I?
I’m a louse fly of a wallglider (an alpine bird)
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