Analytical Tools
Microscopy
Chapter 4
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INTRODUCTION
• Microscopy is applicable to every area of
forensic science
– More than simply looking at small things
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MAGNIFICATION SYSTEMS
• A single lens used to
form an enlarged
image of an object is
a simple
magnification system
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MAGNIFICATION SYSTEMS
• A compound magnification system consists of
two stages of magnification and the total
magnification is the product of the
magnification of the first and second lens
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MAGNIFICATION SYSTEMS
– Observer looks at the first
image with a lens that
produces an enlarged
image called a virtual
image
• The image perceived by the
eye; a real projectable
image does not exist
– I.e. your image in a mirror
– A real image is one that
could be projected onto a
screen
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THE LENS
– A lens is a translucent material that bends light in
a known and predictable manner
– Size and position of an image produced by a lens
can be determined through geometry based on
the focal length of the lens
• Focal length is the distance between two points of
focus on either side of the lens
– Determines much of the image quality
• Resolution is the minimum distance two objects can be
separated and still be seen as two objects
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THE LENS
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THE LENS
– Magnification with one lens is not indefinite
– As magnification increases, lens diameter
decreases to bend the light more to make a larger
image
• Practical limit for simple lens is 10x to 15x
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COMPOUND MAGNIFYING SYSTEMS
– Exceeds the limits imposed by a simple lens
• A second lens is placed in front of the first to further
enlarge the image
• Total magnification of the microscope is the product of
the two lenses
– Lenses of up to 40x can be used in a compound microscope
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COMPOUND MAGNIFYING SYSTEMS
• Lenses in compound microscopes have resolution limits
– Continued magnification of an image is possible, but the
resolution is not improved
» Called empty magnification
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THE MICROSCOPE
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THE MICROSCOPE
– Eyepiece or ocular is the lens that one looks into
when viewing an object microscopically
• A microscope having one eyepiece is monocular
• A microscope having two eyepieces is binocular
– Area seen when looking through the eyepieces is
called the field of view
– Objective lens is closest to the
– object or specimen being studied
• Most important part of microscope
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THE MICROSCOPE
– Numerical aperture is an angular measure of the
lens’ light-gathering ability and its resolving
quality
– Tube length is the distance from the lowest part of
the objective to the upper edge of the eyepiece
• Standardized at 160mm
– Coverslips are thin glass plates that are placed on
top of mounted specimens
• Protects the specimen and objective from damage
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THE MICROSCOPE
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THE MICROSCOPE
– The stage is the platform where the specimen sits
during viewing
• Moved to focus the specimen
– Portion of the specimen in the field of view is sitting in the
same horizontal plane
• May be mechanical, rotating or both
– Mechanical stages have knobs for control of movement
– Rotating stages can spin 360° but not move back and forth
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THE MICROSCOPE
– The condenser is used to obtain a bright, even
field of view and improve image resolution
• Lenses located below the stage that focus or condense
the light onto the specimen field of view
– A condenser diaphragm is used to eliminate excess light and
adjust for contrast in the image
– Field diaphragm allows more or less light into the lens system
of the microscope
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THE MICROSCOPE
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THE MICROSCOPE
– Illumination is critical to a quality image
• Critical and Köhler are the two main types of
illumination used in microscopy
– Critical illumination concentrates light on the
specimen with the condenser lens
» Produces an intense lighting that highlights
edges, but may be uneven
– Köhler illumination sets the light rays parallel
throughout the lens system, allowing them to
evenly illuminate the specimen
» Considered standard
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REFRACTIVE INDEX
– Refraction, or bending of light, is one
characteristic that allows lenses to focus a beam
of light onto a single point
• Occurs when light passes from one medium to another
when there is a difference in the index of refraction
between the two materials
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REFRACTIVE INDEX
• Refractive index is defined as the relative speed
at which light moves through a material with
respect to its speed in a vacuum
– N=C/v
» N is index of refraction, C is speed of light, v is
velocity of light in that material
– Angle of refracted light is dependent on both the
angle of incidence and the composition of the
material into which it is entering
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REFRACTIVE INDEX
– The normal is a line perpendicular to the
boundary between two substances
– Snell’s Law: N1 x sin(q1) = N2 x sin(q2)
• N is refractive indices of material 1 and material 2, q is
angle of light traveling through the material with
respect to the normal
• Mounting media or mountants are used so samples can
be viewed in transmitted light
– Samples must be in a material with a refractive index that is
close to their own
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REFRACTIVE INDEX
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POLARIZED LIGHT MICROSCOPY
• Exploits optical properties of materials to
discover details about the structure and
composition of materials
– Isotropic materials demonstrate the same optical
properties in all directions
• Gases, liquids, certain glasses and crystals
– Anisotropic materials have optical properties that
vary with the orientation of the incoming light and
the optical structure of the material
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POLARIZED LIGHT MICROSCOPY
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POLARIZED LIGHT MICROSCOPY
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POLARIZED LIGHT MICROSCOPY
• PLM uses the fact that anisotropic materials
divide light rays into two parts to yield
information about the material
– A polarizer is a special filter that lets light pass
through only in a “preferred” direction
• Light that vibrates only in one direction is called
polarized light
– The analyzer is a polarizing filter that is aligned
opposite of the polarizer
• Located above the objectives; can manually be slid into
or out of the light path
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POLARIZED LIGHT MICROSCOPY
– Birefringence is the result of the division of light
into at least two rays when it passes through
certain types of material
• Δn= ne - no
– no is the refractive index for the ordinary ray, ne is the
refractive index for the extraordinary ray
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POLARIZED LIGHT MICROSCOPY
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POLARIZED LIGHT MICROSCOPY
– Retardation is difference in velocity of the
ordinary and extraordinary rays
• R=t(n2 – n1)
– r is retardation, t is thickness and n2 – n1 is birefringence
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POLARIZED LIGHT MICROSCOPY
– In the PLM, the out-of-phase waves of light strike
the analyzer and are diffracted into various colors
depending on the wavelengths being added and
subtracted, called interference colors
• Colors are indicative of the fiber’s polymer type and
molecular organization
• Birefringence of a fiber can be determined with PLM
– Numerical difference between the maximum and minimum
refractive indices
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POLARIZED LIGHT MICROSCOPY
• Michel-Levy Chart gives diameter,
birefringence and retardation
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FLUORESCENCE MICROSCOPY
– Fluorescence is the
luminescence of a substance
excited by radiation
• Emission stops when
excitation stops
• Emitted wavelength is longer
than excitation radiation
– Luminescence is subdivided
into phosphorescence,
which is characterized by
long-lived emission, and
fluorescence
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FLUORESCENCE MICROSCOPY
– In a fluorescence microscope, the specimen is
illuminated with light of a short wavelength
• Part of light is absorbed by specimen and reemitted as
fluorescence
• Fluorescence has less energy that exciting radiation
• Fluorophores, or fluorescent components, can be excited
by near UV invisible radiation and seen in the visible range
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FLUORESCENCE MICROSCOPY
– Fluorescence microscope has special light source
and a pair of complementary filters
• Lamp should be rich in short wavelengths
– High-pressure mercury arc lamps
• Primary or excitation filter is placed between lamp and
specimen
• Secondary, barrier, or suppression filter prevents
excitation light from reaching the observer’s eye
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Electron Microscopy
– Electron microscopes employ a particle beam of
electrons focused by magnetic lenses
• Much higher resolving power and greater depth of field
than light microscopes
• Either transmission (TEM) or scanning (SEM)
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Electron Microscopy
• In TEM, the electron
beam passes through
a very thinly sliced
specimen
• In SEM, a beam of
electrons rasters
across a specimen to
provide a non-colored
image of its surface
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Electron Microscopy
» SEM is used to analyze paint, particles, fractures,
toolmarks, and gunshot residue
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CHAPTER SUMMARY
• Microscopy provides fast, low-cost, and
definitive results to the trained scientist
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