JS 112: Microscopy

1. JS 112: Microscopy • I. Microscopy Learning Objectives • Learn the parts of a compound microscope • Define magnification, field of view, working distance and depth of focus • Describe comparison and stereoscopic microscope • Define plane polarized light and how a polarizing microscope works • Define the theory and properties of light • Microspectrophotometry and SEM

2. Forensic Applications of Microscopy • Hair and Fibers • Drugs • Questioned Documents • Glass • Paint • Explosives • Botanicals • Soils

3. Introduction to the Microscope • Optical instrument using a lens or lenses to magnify and resolve fine details of objects • Earliest “microscope” = magnifying glass • Light refracted, or bent resulting in virtual image that can’t be viewed directly that is larger than life • Magnifying glasses yield 5x to 10x mag

4. Anatomy of a compound microscope 1- Base- support or foundation 2- Arm- supports - handle 3- Stage- horizontal plate to hold specimen 4- Body tube – hollow tube on which lenses are mounted 5- Course adjust- focus moving tube 6- Fine adjust-focus moves tubes 7- Illuminator- transmitted if transparent Epiillumination if opaque 8- Condenser-collects light with iris diaphragm to control amount of light- “squinting” 9- Objective-revolving nose piece-parfocal 10-Eyepiece lens – with one-monocular – with two- binocular

5. Compound Microscope • Real, inverted, magnified virtual image-up to 1500X • Each lens is inscribed with mag power. Total mag = power of the objective multiplied by power of eyepiece. • Each objective - numerical aperture (N.A.). The higher the N.A. the better resolved the detail • Field of view- size of specimen area is inversely proportional to the mag power – better to start low • Depth of focus- the amount of the cross section of the specimen (thickness) that is in focus is inversely proportional to the mag power • Optical principles incorporated into 5 light microscopes: • Compound, Comparison, Stereoscopic, Polarizing Micro spectrophotometer

7. Comparison Microscope • Forensic microscopy requires side-by-side comparison of specimens • Two compound microscopes combined into one using a bridge with mirrors and lenses • Two monocular units into a single binocular unit • See a circular image equally divided by a fine line. The left half of the field is from the specimen under the left hand objective and the right half from the specimen under the right hand objective • Comparison scopes designed to compare bullets, cartridges and other opaque objects come with vertical or reflected illumination.

8. Virtual comparison microscope and firearms evidence sitehttp://www.firearmsid.com/

9. Forensic microscopy of hair may reveal important information • Shed versus forcibly removed hairs • Reference: http://www.fbi.gov/hq/lab/fsc/backissu/july2000/deedric1.htm#Hair%20Evidence

10. Stereoscopic Microscope • Details of physical evidence do not always need high mag examinations • Stereoscopic microscopes are quite adequate with 10x to 125x • Distinct 3D image- actually 2 monocular microscopes • Right side up images using prisms vs. compound inverted and reversed • Most frequently used in crime labs • Wide field of view and great depth of focus ideal to locate trace evidence • Large working distance good for large items • Paint, soil, gunpowder residue, marijuana

11. Spectrophotometry- Theory of Light-1Seehttp://micro.magnet.fsu.edu/primer/lightandcolor/index.html for additional information and review • White light is composed of a range of colors • ROYGBIV • Red glass absorbs all component colors except red passing through or transmitted • Color is a visual indication of an objects ability to absorbing some and reflect other components of visible light

12. Spectrophotometry- Theory of Light -2 • Light described as continuous wave or stream of discrete energy particles • Light as a wave in up and down motions has wavelength (lambda-) in nanometers • The frequency is the number of crests per unit time • Speed of light is 300 million m/s- symbol c • Frequency and wavelength are inversely proportional • F=c/lambda

13. Spectrophotometry- Theory of Light-3 • Visible light is only a small part of the entire electromagnetic spectrum • Normal light is a collection of waves possessing a range of wavelengths and are out of step with each other • When pulsating in unison they are coherent or laser –Light amplification by the simulated emission of radiation

14. Spectrophotometry- Theory of Light-4 • Absorption of Electromagnetic Radiation • Any substance exhibit selective absorption of electromagnetic radiation • Different materials have different absorptions • The selective absorption of a substance is measured by an instrument called a spectrophotometer. • It produces an absorption spectrum depicting the absorption of light as a function of wavelength or frequency. Absorption of UV, visible and IR are particularly applicable for identification of organic substances- How much- Beer’s Law- A=kc • A= absorption c=concentration k=proportionality

15. Spectrophotometer • Instrument used to measure and record the absorption spectrum of a chemical substance • Components- 1. Radiation source • 1- Radiation source (UV, vis, IR) • 2. Monochromator or frequency selector • 3. Sample holder • 4. Detection to convert electromagnetic radiation into an electric signal (digitizer) • 5. Recorder

16. Spectrum • Different materials always have distinctively different IR spectra • Each IR spectra is equivalent to a “fingerprint” of that substance and no other

17. Microspectrophotmetry • Microscope magnifies images to search and locate traces of physical evidence characterize morphology • Linked to a computerized spectrophotometer • Ideal marriage- view a particle under a microscope and focus a beam of light to obtain an absorption spectrum- Visible or IR • Characterization of trace quantities of evidence • Also see Applied Spectral Imaging for using CCD with spectrometry

18. Scanning Electron Microscope • Beam of electrons from hot tungsten filament focused with electromagnets on the specimen • Causes emission of electrons • Electrons are collected (secondary and backscatter) and amplified on a TV! • Convert emitted electrons into an image • High mag, high resolution and great depth of focus • Mag range of 10X to 100,000 • Depth some 300 times better than optical systems at similar mags • X rays can be used - elemental composition with SEM

19. Pollen and spore SEM imageshttp://www.cci.ca.gov/Reference/Pollen/pollen.htm

20. Summary • A microscope is an optical instrument that uses a lens or a combination of lenses to magnify and resolve the fine details • Many different microscopes are used by forensic scientists • Compound- object to be examined is placed under the lower lens called the objective and the magnified image is viewed through the upper lens called the eyepiece • Side by side comparisons can be accomplished using the comparison microscope consisting of two independent objective lenses joined by an optical bride to a common eyepiece lens. Modern firearms examination began with the introduction of the Comparison microsc • Stereoscopic microscopy is two monocular compound microscopes properly space and aligned to present a 3D image. Large working distance, large field of view and depth of focus • Microspectrophotometers couples microscopy to spec so a specimen under a microscope can simultaneously have absorption spectrum • SEM uses electrons to produce a highly magnified image from 10-100,000x and can be combined with Xray to characterize elements