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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.

• 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

• Transmitted illumination: light that passes

up from the condenser and though the

specimen

• 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 Comparison Microscope

Polarizing Microscope Stereoscopic Microscope

Cell division in a frog's egg.

Microspectrophotometer Scanning Electron Microscope (SEM)

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SEM Data

Nanoscaled polyimide structures

Side-wall morphology of solar cell gridline

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

– 5x smaller than the smallest object that can be seen directly by the naked eye

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

Lenses

Refraction of a light ray as it passes through a prism

Lenses

• Light passing through two “identical” prisms stacked base to base would intersect at point I– produce a real image

– converging lens

Focal Point & Focal Length

• 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

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Simple Magnifier

• 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

The Compound Microscope

• 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

– specimen on left scope on left side of field

– specimen on right scope on right side of field

The Comparison Microscope

• Bullet comparisons

• Hair & Fiber comparisons

• Questioned documents

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Test Fire Reference Gun Use A Comparison Microscope

Striations match

Stereoscopic Microscope

• Two separate

monocular

microscopes

• Each has its own set of

lenses

Stereoscopic Microscope

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

ObjectsScanning 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 preparedThe sample is placed inside the microscope's vacuum

column through an air-tight door

• 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

• Near the bottom, a set

of scanning coils

moves the focused

beam back and forth

across the specimen,

row by row

• 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 final image is

built up from the

number of electrons

emitted from each spot

on the sample

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Fiber AnalysisEnergy-Dispersive X-Ray

Analysis (EDX)• Electron beam ~5,000-

20,000eV

• 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)