Chapter 4 Methods for Examining Biological Evidence Prof. J. T. Spencer Adjunct Prof. T. L. Meeks

Learning Goals and Objectives Biological evidence forms a critical core of forensic investigations. Accurate observations and measurements are key to analyzing these data…

Learning Goals and Objectives How measurements are taken - units, accuracy and precision of measurements Limitations of our senses on our interpretation of the world around us What is electromagnetic radiation and how do we perceive it What is the SI system of measurement and how does it work How the uncertainty of a measurement is estimated and indicated

Learning Goals and Objectives What is meant by the accuracy and precision of a measurement How a lens works to create a magnified image What are the basic principles of microscope operation That is meant by resolution, magnification, numerical aperture, and related terms

Learning Goals and Objectives What are the main types of optical microscopy and how do they work How electron microscopy works What other types of microscopy are available and when they are used

CHE Matter; Measurement It probably comes as no surprise that accurate, reliable and meaningful observations and measurements are fundamental to all of forensic science. These observations can be either quantitative, involving detailed measurement, and qualitative, involving careful descriptions.

CHE Systems Systems Metric - base 10 Metric - base 10 SI- international scientific system SI- international scientific system – massKilogram – lengthMeter – timeSecond – electric currentAmpere – temperatureKelvin – lightLumen (Candela) – amountMole Factor label method for conversions Factor label method for conversions Measurement

CHE Prefixes Prefixes MegaM10 6 Kilok10 3 Decid10 -1 Centic10 -2 Millim10 -3 Micro  Nanon10 -9 Measurement

CHE Matter; Measurement A B Which is True? A = B A > B A < B

CHE Matter; Measurement A B Which is True? A = B A > B A < B

CHE Matter; Measurement A B Which is True? A = B A > B A < B

CHE Uncertainty in Measurement Precision and Accuracy Precision and Accuracy Precision - how closely individual measurements agree Precision - how closely individual measurements agree Accuracy- how closely the measurements agree with the true value Accuracy- how closely the measurements agree with the true value Significant Figures Significant Figures All measurements are inaccurate intrinsically All measurements are inaccurate intrinsically measured quantities are reported such that the last figure is uncertain measured quantities are reported such that the last figure is uncertain

CHE Good Precision Poor Accuracy Good Precision Good Accuracy Poor Precision Poor Accuracy Uncertainty in Measurement

CHE Accuracy in Measurement

Tools for Understanding Biological Evidence When doing observations and measurements, we perceive the world only indirectly through the facility of our senses of sight, touch, taste, hearing and smell…

The Microscope Provides a direct image of a small object of interest 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 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 A typical animal cell is nm in diameter 5x smaller than the smallest object that can be seen directly by the naked eye 5x smaller than the smallest object that can be seen directly by the naked eye

The Microscope Produce a magnified image of a specimen Produce a magnified image of a specimen Separate the details in the image Separate the details in the image Render the details visible to the human eye or camera Render the details visible to the human eye or camera

Microscopy History

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 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 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 The distance of this point from the lens is the focal length

Simple Magnifier Object O is placed close to the lens Object O is placed close to the lens rays converge but do not intersect rays converge but do not intersect real image not formed real image not formed The observer’s eye follows rays back to the point of apparent origin (I) The observer’s eye follows rays back to the point of apparent origin (I) (I) bigger than object (I) bigger than object

The Compound Microscope Rays pass first through the objective lens forming a real, slightly enlarged, inverted image Rays pass first through the objective lens forming a real, slightly enlarged, inverted image The second lens (eyepiece) acts as a simple magnifier The second lens (eyepiece) acts as a simple magnifier

Compound Microscope Both lenses produce magnification Both lenses produce magnification Overall magnification is found by multiplying the two magnifications Overall magnification is found by multiplying the two magnifications Magnification determined mainly by objective Magnification determined mainly by objective

Limitations of Light Microscope Radiation of a given wavelength can’t be used to probe structural details much smaller than its own wavelength Radiation of a given wavelength can’t be used to probe structural details much smaller than its own wavelength Light Microscope Light Microscope limited to range of visible light limited to range of visible light 0.4 mm (violet) to 0.7 mm (deep red) 0.4 mm (violet) to 0.7 mm (deep red) bacteria & mitochondria (~0.5mm wide) smallest objects that can be seen clearly bacteria & mitochondria (~0.5mm wide) smallest objects that can be seen clearly

The Comparison Microscope Two compound microscopes combined into one unit Two compound microscopes combined into one unit When viewer looks through the eyepiece, a field divided into two equal parts is observed 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 left scope on left side of field specimen on right scope on right side of field specimen on right scope on right side of field

The Comparison Microscope Bullet comparisons Bullet comparisons Hair & Fiber comparisons Hair & Fiber comparisons Questioned documents Questioned documents

Test Fire Reference Gun

Use A Comparison Microscope

Striations match

Phase-Contrast Microscope A phase-contrast microscope splits a beam of light into 2 types of light, direct and refracted (reflected) and brings them together to form an image of the specimen. A phase-contrast microscope splits a beam of light into 2 types of light, direct and refracted (reflected) and brings them together to form an image of the specimen. Where they are "in-phase" the image is brighter, where they are "out of phase" the image is darker, and by amplifying these differences in the light, it enhances contrast Where they are "in-phase" the image is brighter, where they are "out of phase" the image is darker, and by amplifying these differences in the light, it enhances contrast It allows the detailed observation of living organisms, especially the internal structures. It allows the detailed observation of living organisms, especially the internal structures.

Light confined to a single plane of oscillation is said to be polarized. Light confined to a single plane of oscillation is said to be polarized. Many crystals are birefringent (light is refracted into 2 separate rays). Many crystals are birefringent (light is refracted into 2 separate rays). Reduces glare by transmitting light in a vertical plane only. Reduces glare by transmitting light in a vertical plane only. Polarizing Microscope

Why do molecules rotate polarized light? A molecule possessing nonsupeposable mirror images is termed chiral. Naturally occurring substances are often found as just one mirror image (enantiomer). Enantiomers are identical in physical properties and identical chemical properties when they react with nonchiral reagents; only in a chiral environment will the differences show. Enantiomers rotate of polarized light in different directions.

Optical Activity Non-superimposable mirror images Non-superimposable mirror images Mirror Left Hand Right Hand

Optical Isomers Amino Acids are Chiral Amino Acids are Chiral Mirror Plane Enantiomers alanine

Polarizing Potato Starch

In fluorescence microscopy, specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet (or near- ultraviolet) light source. In fluorescence microscopy, specimens are first stained with fluorochromes and then viewed through a compound microscope by using an ultraviolet (or near- ultraviolet) light source. The microorganisms appear as bright objects against a dark background. The microorganisms appear as bright objects against a dark background. Fluorescence microscopy is used primarily in a diagnostic procedure called fluorescent- antibody (FA) technique, or immunofluorescence. Fluorescence microscopy is used primarily in a diagnostic procedure called fluorescent- antibody (FA) technique, or immunofluorescence. This technique is especially useful in diagnosing syphilis & rabies. This technique is especially useful in diagnosing syphilis & rabies. Fluorescence Microscope

Hand section of sugarcane vascular bundle viewed with fluorescence microscope Sugarcane vascular bundle viewed with traditional staining and transmitted light (Bright Field) microscopy Hand-section of Sugarcane stem with a vascular bundle Stained with Toluidine Blue & Viewed with Bright Field microscopy

An egg that was stained with a dye that appears yellow in a fluorescence microscope. An unfertilized egg stained with a dye that appears red in a fluorescence microscope. The line indicates size (10 micrometers = 1/100 of a millimeter). The egg is about 100 micrometers in diameter, so about ten eggs would fit side by side on a pencil line.

Stereoscopic Microscope Two separate monocular microscopes Two separate monocular microscopes Each has its own set of lenses Each has its own set of lenses Gives a 3D view Offers the largest stage for evidence

Stereoscopic Microscope Using the Stereo MicroscopeUsing the Compound Microscope

Stereoscopic Microscope Cell division in a frog's egg.

FT-IR Microspectrophotometer

Photocopier Toner Analysis important for establishing corroborative evidence linking documents to specific locations in forensic investigations of corporate crime important for establishing corroborative evidence linking documents to specific locations in forensic investigations of corporate crime Must be performed non-destructively Must be performed non-destructively can’t remove toner from paper can’t remove toner from paper physical size of specimen is very small physical size of specimen is very small microscope to find sample microscope to find sample FT-IR to analyze the sample FT-IR to analyze the sample

Photocopier Toner Analysis

Scanning Electron Microscope This scanning electron microscope has a magnification range from 15x to 200,000x and a resolution of 5 nanometers This scanning electron microscope has a magnification range from 15x to 200,000x and a resolution of 5 nanometers

Range of Readily Resolvable Objects

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 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. 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 Because the SEM illuminates them with electrons, they also have to be made to conduct electricity

How do you make a mosquito conductive? 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 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 Air is pumped out of the column An electron gun [at the top] emits a beam of high energy electrons. 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 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. 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 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 The final image is built up from the number of electrons emitted from each spot on the sample

TEM Pictures Silver Nanoprisms Gold Nanoparticle

Fiber Analysis

Who am I?

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