The Microscope. Utilizing a microspectrophotometer for examining trace physical evidence.

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1 The Microscope Utilizing a microspectrophotometer for examining trace physical evidence. 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. 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 1

2 Compound Microscope Comparison Microscope Polarizing Microscope Stereoscopic Microscope Cell division in a frog's egg. Microspectrophotometer Scanning Electron Microscope (SEM) 2

3 SEM Data The Microscope Nanoscaled polyimide structures Side-wall morphology of solar cell gridline 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 nm in diameter 5x smaller than the smallest object that can be seen directly by the naked eye Produce a magnified image of a specimen Separate the details in the image Render the details visible to the human eye or camera The Microscope 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 produce a real image converging lens 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 3

4 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 Simple Magnifier 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 4

5 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 5

6 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 6

7 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 7

8 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 8

9 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 9

10 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 Who am I? 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) 10