this presentation deals with the introduction of some of the commonly used optical microscopes in forensic labs; compound microscope, stereoscopic microscope, comparison microscope, fluorescence microscope and polarized microscope.

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•Instrument that uses a lens or a combination of lenses
to magnify and resolve the fine details of an object.
•Early methods for examining physical evidence relied
solely on the microscope.
The Microscope

3. Vocabulary
• Magnification – larger
image
• Resolution – clearer image
• Numerical Aperture – light
gathering capacity of a lens
• Working Distance – the
distance from the bottom
of an objective to the in-
focus area of an object
(distance between
specimen and lens)
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4. Magnification
• Magnification is the enlargement of the image
• The magnification of a microscope is given by-
• Generally used class microscope has following
magnification-
Mmicroscope = Moccular X Meyepiece
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5. Resolution
• Resolution is defined as the
ability to distinguish two very
small and closely-spaced
objects as separate entities.
• Resolution is best when the
distance separating the two
tiny objects is small.
• Degree to which detail in
specimen is retained in
magnified image.
• Resolving power-
– Unaided eye – 0.1 mm apart
– Microscope - 0.2 µm apart
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6. Numerical Aperture
•NA is light gathering capacity of objective
•Limit of resolution = 0.61λ
NA
• NA (Numerical Aperture) = n sinα
Wavelength of
illumination
Aperture angle
Refractive index of air or liquid
between specimen and lens
•The N.A. of each objective lens is inscribed in the metal tube, and ranges from
0.25-1.4
•The higher the N.A., the better the light-gathering properties of the lens, and the
better the resolution.
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7. Simple Microscope
• Similar to a
magnifying glass
and has only one
lens.
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8. Compound Microscope
• Compound microscope
was constructed by
Robert Hooke (1665) & is
forerunner of present day
compound microscope.
– Most widely used
microscope
– Light passes through 2
lenses
– Can magnify up to 2000x
•Early Compound Microscopes
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9. Compound Microscope
https://www.youtube.com/watch?v=RKA8_mif6-E
• A compound microscope consists of various components which gather
light and redirects the light path so that a magnified image of the
viewed object can be focused within a short distance.
I. Light source – source of illumination
II. Condenser – collimates the light
III. Sample Stage – specimen is placed over this
IV. Objective lens – produces a real intermediate image onto the ocular
front plane
V. Oculars – re-focus the intermediate image on the retina as a larger
virtual image
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10. Principle of Compound Microscope
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Original text
As seen through a compound
microscope
•This reversal is always seen
using a standard compound
microscope.
•It's the reason when we move
a slide right the image moves
left, and
•when we move a slide
downward the image moves
upward.

11. The specimen
Final image
Intermediate
image
Eye
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12. Modern Compound Microscope
The microscope is consists of:
•mechanical system which
supports the microscope,
•an optical system which
illuminates the object under
investigation
•light passes through a series of
lens to form an image of the
specimen.
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13. Comparison Microscope
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14. A - B - Two identical microscopes
C -C' - Specimens to compare
D - Comparison eyepiece (optical bridge)
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Unlike any other microscope,
it looks at two different
objects at the same time. As
its name implies, it is used to
compare objects
Youtube video:
https://www.youtube.com/watch?v=Ci1Qi3Ire_E

15. Comparison Microscope
• The comparison microscope consists of two
independent objective lenses joined together by
an optical bridge to a common eyepiece lens.
• When a viewer looks through the eyepiece lens of
the comparison microscope, the objects under
investigation are observed side-by-side in a
circular field that is equally divided into two parts.
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16. Bullet markings Photographed
using Comparison Microscope
• Modern firearms examination began with the
introduction of the comparison microscope, with its
ability to give the firearms examiner a side by side
magnified view of bullets.
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17. Forensic Applications
1. It enables side by side comparison of the rifling impressions on projectile found at the
crime scene with a test projectile fired in the laboratory.
2. Similar principle is used for comparison of cartridge cases, where we compare
a) firing pin marks (formed when a pin hits the primer and makes the cartridge
explode),
b) breech face marks (the impression made when the cartridge is pressed against the
end of the barrel during the explosion) and
c) ejector/extractor marks (caused when the cartridge is discharged or ejected from
the barrel).
3. The same principle is used to compare the tool marks such as- screwdriver, saw, saw
edged knife, axe, dagger etc.
4. Comparison microscope is used for looking at adhesive strips from a letter bomb parcel,
and comparing them with a roll of adhesive tape found at a potential perpetrator’s.
5. Another area of use of comparison microscopy is the examination of traces from cars
such as glass and artificial glass splinters, paint traces etc.
6. Used for comparison of hairs and fibres of different origins.
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18. Stereoscopic Microscope
• The stereo or stereoscopic microscope is an optical microscope variant designed for low
magnification observation of a sample, typically using light reflected from the surface of an
object rather than transmitted through it.
• The instrument uses two separate optical paths with two objectives and eyepieces to
provide slightly different viewing angles to the left and right eyes.
• This arrangement produces a three-dimensional visualization of the sample being
examined.
• Reflected light illumination rather than transmitted illumination (Unlike a compound light
microscope).
• Light reflected from the surface of an object rather than light transmitted through an
object.
• Use of reflected light from the object allows examination of specimens that would be too
thick or otherwise opaque for compound microscopy.
• Stereomicroscopy overlaps macrophotography for recording and examining solid samples
with complex surface topography, where a three-dimensional view is needed for analyzing
the detail.
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The stereoscopic microscope is actually
two monocular compound microscopes
properly spaced and aligned to present a
three dimensional image of a specimen to
the viewer, who looks through both
eyepiece lenses.
Advantage:
•Great working distance
•Enhanced depth of field
•Ease of sample manipulation on
stage
•Larger samples can be analyzed
•3-D view of image just like human
eyes
Disadvantage:
•Low magnification
•Low resolution

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Fig: Details of a suspected document pen
over toner.
Fig: Detail of a bank note (embossed elements).

21. Forensic Applications
• Most important application of Stereoscopic Microscopy is in the
fields of Questioned Document Examination.
– It is especially adapted for the examination of inks, colors, erasures,
changes, interlineations, and overwriting.
– For the comparison of disturbed and undisturbed paper surfaces, pen,
and pencil points, the tint,
– Texture and condition of paper surfaces,
– The texture and quality of typewriter ribbons, written and printed
characters, and type faces.
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22. Fluorescence Microscope
https://www.youtube.com/watch?v=PCJ13LjncMc
• Excites and observe fluorescent molecules.
• A fluorescence microscope is an optical microscope
that uses fluorescence and phosphorescence instead
of, or in addition to, reflection and absorption to study
properties of organic or inorganic substances.
• The "fluorescence microscope" refers to any
microscope that uses fluorescence to generate an
image.
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23. Fluorescence Microscopy
• 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 auto-
fluorescence image, based on their chemical makeup.
• Specimens usually stained with fluorochromes.
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24. Excited state
Ground state
excitation
Shorter wavelength,
higher energy
emission
longer wavelength, less
energy
•Exposes specimen to ultraviolet,
violet, or blue light
•Shows a bright image of the object
resulting from the fluorescent light
emitted by the specimen
(fluorophores).
Fluorophores:
•Different fluorescent dyes can be used to stain
different structures or chemical compounds.
•Examples of commonly used fluorophores are
fluorescein or rhodamine.
•An ideal fluorescent image shows only the
structure of interest that was labelled with the
fluorescent dye.
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26. lamp
sample
camera
Emission filter
Dichroic mirror
Excitation filter
Emission filter
Transmission
(%)
wave length (nm)
Excitation filter
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28. Sample
Objective lens
Excitation light
Tube lens
Emission light
Pinhole
Detector
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29. Fluorescence
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30. Uses
1. To study the membrane dynamics (endocytosis, receptor bindings
etc.)
2. To measure the concentration of Ca+2 ions, pH changes and protein
interactions.
3. Determine the localisation of specific (multiple) proteins
4. Determine the shape of organs, cells, intracellular structures
5. Examine the dynamics of proteins
6. Study protein interactions or protein conformation
7. Examine the ion concetration etc.
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31. Polarized Light Microscope
• Polarized light microscopy is a techniques involving polarized light
for illumination of the sample, while blocking the directly
transmitted light with a polariser orientated at 90 degrees to the
illumination.
• Polarized light microscope is designed to observe specimens that
are visible primarily due to their optically anisotropic character
(birefringent).
• The microscope must be equipped with both a polarizer, positioned
in the light path somewhere before the specimen, and
• An analyzer (a second polarizer), placed in the optical pathway
between the objective rear aperture and the observation tubes or
camera port.
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32. When the electric field vectors of light are restricted to a single plane
by filtration, then the light is said to be polarized with respect to the
direction of propagation and all waves vibrate in the same plane.
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33. Fig- 1
Fig- 2
Fig- 3
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34. Polarizers
• Polarizers specifically transmit one polarization
angle of light
• Crossed polarizers transmit no light
X
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36. Analyzer (upper polarizer) -- a polarizing prism
located above the microscope stage, between
the objective lens and the eyepiece.
This restricts the transmission of light vibrating
perpendicular to the polarizer. The analyzer
can be slipped in or out of the light path or
rotated for partially crossed polarized light.
Light passing through the polarizer will not
pass through the analyzer unless the vibration
direction of the light is changed between the
two prisms.
Polarizer (lower polarizer) -- a polarizing prism
located beneath the microscope stage
(between the light source and the object of
study).
This restricts transmission of light to that
vibrating in only one (N-S) direction.
Some microscopes have a different orientation
direction.
In effect, it plane polarizes the incident light
beam.
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37. Uses of Polarized Microscopy
• Polarizing microscopy has found wide
applications for the study of birefringent
materials; materials that split a beam of light
in two, each with its own refractive index
value.
• The determination of these refractive index
data provides information that helps to
identify minerals present in a soil sample or
the identity of a man-made fiber.
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