MICROSCOPY
Micro means small
Skopein means to
see
Microscopes
Preparation of
specimens for light
microscopy
The Instruments

Bright-field Microscope
Compound
 Comparison
 Stereoscopic
 Microspectrophotometer


Scanning Electron Microscope
HOW MICROSCOPES WORK
Microscopy History
•Dutch spectacle-makers (1590), Janssens,
discovered that nearby objects appeared
greatly enlarged with lenses.
•Galileo (late 1600s), based on the Janssens
experiments, worked out a
much better instrument
with a focusing device.
Janssen
Galileo
Microscopy History
•Later Microscopes
Olympus
(modern)
Pacino, 1870
COMPOUND LIGHT MICROSCOPE



The most common microscope compound light microscope (LM).
Two sets of lenses: ocular and
objective.
The total magnification: multiply
magnification of the objective lens
with the ocular lens.




e.g., ocular is 10x and the objectives is
100, total mag. will be 1000x.
Optical system comprised of
condenser, objective lens, eyepiece
lens and illuminator.
The compound light microscope uses
visible light. ( = 400 – 700 nm).
Virtual image is any specimen
viewed through a lens. 6
COMPOUND MICROSCOPE
1. Base
2. Arm
3. Stage
4. Body tube
5. Coarse adjust
6. Fine adjust
7. Illuminator
8. Condenser
9. Objective lens
10. Occular lens
ELECTROMAGNETIC SPECTRUM
COMPARISON MICROSCOPE
Two compound microscopes
combined into one unit
 When viewer looks through
the eyepiece, a field divided
into two equal parts is
observed

Firearms Identification:
Bullet comparisons
 Hair & Fiber comparisons
 Questioned documents

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COMPARISON MICROSCOPE
Split-image
comparison of firing
pin
imprints in coaxial
incident light
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COMPARISON MICROSCOPE
Split-image comparison of banknotes:
on the left the original, on the right the forgery
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STEREOMICROSCOPE
Also called the dissecting microscope
 Working distance from objective lens to specimen.
 Examiner can manipulate the specimen

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Stereoscopic Microscope
• most widely used microscope in crime laboratories
• most versatile of the microscopes used
• A binocular microscope.
Advantages:
• large working distance for bulky samples
• three-dimensional image
• image that is not inverted or reversed
Disadvantage:
• magnifying power of only 10-125X
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ILLUMINATION
Vertical Illumination (ex. stereomicroscope)
• Illumination from above the specimen
• Used when studying opaque samples
• Light is reflected off the specimen’s surface into the lens
Transmitted Illumination (ex. compound microscope)
• Used only with transparent specimen
• Light is directed up through the specimen from the base.
Microscope Characteristics
Field of View = the span of the area in view
Depth of Focus = the thickness of the region in focus
Field of view and depth of focus both decrease as
magnifying power increases.
• High powers of magnification “sacrifice” both the size of the area in
view and the thickness of the region in focus
• Low powers of magnification “sacrifice” detail within the region
under examination and “in focus.”
MICROSPECTROPHOTOMETERS
• Available as visible or infrared spectrometers.
• Details of the sample may be viewed directly.
• The visible or infrared spectrum of the specimen being viewed
may be obtained at the same time.
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One genuine and one
counterfeit $50 bill
Inked line on bill
Visible spectrum of each
ELECTRON MICROSCOPE
A beam of electrons, instead
of light, is used
 Can magnify greater because
the wavelengths of electrons
are much smaller than those
of visible light = 0.005 nm as
opposed to 500 nm
 The best compound light
microscopes can magnify
2000x, electron microscopes
can magnify up to 100,000x
 2 types: TEM & SEM

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SCANNING ELECTRON MICROSCOPY (SEM)

3-D views of the surfaces by aiming a beam of
electrons onto the specimen.
Electrons are bounced off the surface of the
specimen and form a 3D image that is
stereoscopic in appearance.
 Magnification: 1000-100,000x and Depth of
Field very high.
 Can be used to identify the elements present in
the specimen under examination.

SCANNING ELECTRON MICROSCOPE (SEM):
SAMPLE CHARACTERISTICS
The sample must not be “soft.”
“Soft” samples are destroyed by the electron beam.
The sample will emit X-rays characteristic of the elements present
at the surface of the sample. The sample composition may be
analyzed while the sample is being “viewed.”
SEM IMAGES
Human Hair (1100X)
Diatom
SEM IMAGES
Semiconductor Chip
(600X)
SEM IMAGES
Bread Mold (200X)