Ch 7 – 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 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

Compound Microscope

Comparison Microscope

Polarizing Microscope

Stereoscopic Microscope Cell division in a frog's egg.

Microspectrophotometer

Scanning Electron Microscope (SEM)

SEM Data Nanoscaled polyimide structures Side-wall morphology of solar cell gridline

Viewing small Specimens Ch. 7 - Microscopy Viewing small Specimens

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

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

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

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

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

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

The specimen is now prepared

The 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

Fiber Analysis

Energy-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

I’m a louse fly of a wallglider (an alpine bird) Who am I? I’m a louse fly of a wallglider (an alpine bird)