The Microscope. Utilizing a microspectrophotometer for examining trace physical evidence

The Microscope • Compound microscope. • Magnification, field of view, working distance, and depth of focus. • Comparison microscope. • Advantages of s...
Author: Shannon Johnson
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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.

• 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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