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

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

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

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

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

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.

Virtual comparison microscope and firearms evidence site http://www

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

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

Spectrophotometry- Theory of Light-1 See http://micro. magnet. fsu Spectrophotometry- Theory of Light-1 See http://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

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

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

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

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

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

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

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

Pollen and spore SEM images http://www. cci. ca

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