Introduction The applications of microscopy in the forensic sciences are almost limitless. This is due in large measure to the ability of microscopes to detect, resolve and image the smallest items of evidence, often without alteration or destruction. As a result, microscopes have become nearly indispensable in all forensic disciplines involving the natural sciences. Thus, a firearms examiner comparing a bullet, a trace evidence specialist identifying and comparing fibers, hairs, soils or dust, a document examiner studying ink line crossings or paper fibers, and a serologist scrutinizing a bloodstain, all rely on microscopes, in spite of the fact that each may use them in different ways and for different purposes. The principal purpose of any microscope is to form an enlarged image of a small object. As the image is more greatly magnified, the concern then becomes resolution; the ability to see increasingly fine details as the magnification is increased. For most observers, the ability to see fine details of an item of evidence at a convenient magnification, is sufficient. For many items, such as ink lines, bloodstains or bullets, no treatment is required and the evidence may typically be studied directly under the appropriate microscope without any form of sample preparation. For other types of evidence, particularly traces of particulate matter, sample preparation before the microscopical examination begins is often essential. Types of Microscopes Used in the Forensic Sciences A variety of microscopes are used in any modern forensic science laboratory. Most of these are light microscopes which use photons to form images, but electron microscopes, particularly the scanning electron microscope (SEM), are finding applications in larger, full service laboratories because of their wide range of magnification, high resolving power and ability to perform elemental analyses when equipped with an energy or wavelength dispersive X-ray spectrometer.

1. Notes for cbse net jrf
Forensic science
By – priya tamang

2.  It is the most efficient image-processing system available to
date.
 All the appliances provided by technology are no match for the
eye as regards speed and resolution.
 The structure of the eye is related to that of a camera.
 Together with the muscle-adjusted lens (1a), the curved surface
of the cornea (1) projects an optical image onto the retina (2).
The amount of incident brightness is controlled via the variable
diameter of an iris (3).
 A sharp image is provided by the flexible lens, the focal length of
which is changed by muscles in such a way that focusing is
possible on any subject at a distance between approx. 20 cm and
infinity.
 The image itself is picked up on the retina by approx. 130
million receptor rods (recognition of grey levels) and 7 million
cones (color recognition) and transferred to the brain on the
shortest possible path via the optic nerve.

3.  The light rays shown in the following illustration form a viewing
angle of 30°. The following objects, for example, are seen under
this angle:
 The spire of the Ulm Minster with a height of 161 m, seen from
a distance of 300 m
 a photo with a height of 13 cm seen from a distance of 25 cm.
 The details we want to see have a diameter of only 1/100 or
even 1/1000 of a millimeter.
 However, we are unable to go any closer to the object than
approx. 20 cm. As a consequence, the viewing angle becomes
extremely small, which is why we are unable to recognize any
details.
 A similar situation is experienced if we view the spire already
mentioned from a distance of 300 m. The many intricate details
created by the stonemasons cannot be recognized from such
great a distance because the viewing angles are too small.

4.  A remedy has been known for such cases for
centuries: the use of a “magnifying glass”
which – when put between the eye and the
object – makes everything appear larger.
 However, there is a limit to this method: a
magnification of more than 8-fold or 10-fold
is not possible. Anyone who wants to see
more must use the “compound” microscope.

5.  If one lens is not sufficient, several lenses can be arranged one
behind the other. The magnifying effect is thus multiplied,
allowing magnifications of up to 2000x.
 The classic microscope magnifies in two steps: The objective
produces a magnified image of the object in the so-called
intermediate image plane, and the eyepiece or ocular (Latin:
oculus = eye) magnifies the intermediate image in the same way
as a magnifier.
 The beam path on the right shows how light is emitted from an
object ( ← ) and processed in the three lenses. We shall only look
at the rays originating in the two ends of the object.
 This will be sufficient to explain the process of magnification.
The illustration shows the ICS principle (ICS=Infinity Color
corrected System) also used with the Axio lab microscope.
 A further step is included in this modern microscope featuring
“infinity optics”: a tube lens is added to support the objective.

6.  The objective projects an
image at an “infinite”
distance, the tube lens
with its focal length f =
164.5 mm then forms the
intermediate image from
these parallel beams.
 First, let us assume that
nothing decisive for the
image formation happens
in the space between
objective and tube lens.
 The light rays coming
from the focused
specimen plane are
parallel in this space
anyway.
 The eyepiece, in turn,
serves as a magnifying
glass to make this small
intermediate image appear
even more magnified to
the eye.

8.  The optical microscope is used extensively in pharmaceutical
development with the primary application being solid-state analysis.
 The applications range from simple images of drug substance to
illustrate particle size and shape to full optical crystallography.
 The range of utility of the microscope is considerably extended by the
use of polarized light which allows us to obtain crystallographic data on
small individual crystals.
 I will use the term Polarized Light Microscopy (PLM) for all light
microscopy discussions in this chapter.
 Polarized light microscopy provides us a unique window into the internal
structure of crystals and at the same time is aesthetically pleasing due to
the colors and shapes of the crystals.
 The use of PLM as a tool for crystallography extends back at least 200
years. For many of those years, it was the prime tool for examining the
crystal properties of minerals and inorganic chemicals, as well as
organics.
 Pasteur’s seminal studies in the handedness of organic chemicals were
initially conducted on an optical microscope.
 Needham (1958) provides some information on the development of PLM
but frankly I am not aware of any good articles or books that detail the
history of both the development of the polarizing light microscope and
its applications.

9.  The use of polarized light on the optical microscope
allows us to determine the optical crystallographic
properties of the crystal. Optical crystallography is related
to but different from X-ray crystallography.
 Each technique provides unique information about the
crystal structure and the combination of the two is
powerful indeed.
 For example, optical crystallography uniquely yields the
sign and magnitude of the angle between optic axes in a
biaxial crystal.
 This value, among others, can be used for the definitive
identification of the solid-state form.
 This determination can be made on crystals as small as
~5 mm and can be done in a mixture of particles of
different forms. PLM was originally developed for studies
in mineralogy and petrography.
 It has since expanded so that virtually every field that
deals with crystalline materials uses the techniques of
optical crystallography.
 Optical crystallographic measurements can yield as many
as 20 different characteristics, many of which are
numerical.

10.  This set of values is unique for each solid-state
form.
 A hydrate will have different optical
crystallographic values than its anhydrate and
different polymorphs of the anhydrate (or
hydrate for that matter) will have different optical
properties.
 Optical crystallography, then, is a superb tool for
the in situ identification of different solid-state
forms.
 There are two caveats:
 First, we must have good reference data for each
of the forms;
 second, the microscopist must be skilled in the
art and science of optical crystallography.
 It seems to me that the decline in the use of this
science is related to both of these requirements.

11.  There are a number of excellent texts on light and optics in relation to
optical microscopy.
 Much of the discussion below follows Slayter and Slayter (1992),
McCrone et al. (1984), and Needham (1958).
 Gage (1943) and Martin (1966) provide a more detailed and extensive
discussion of light and optics as they relate to microscopy.
 Born and Wolf (1980) is the standard for optics.
 Visible light is a narrow part of the electromagnetic (EM) spectrum
usually considered to extend from 400 to 700 nm (10−9 m) in
wavelength. Wavelengths of the entire electromagnetic spectrum extend
from 10−16 m for gamma rays to 1010 m for long radio waves.
 The spectrum is divided into the following categories based on short to
long wavelength (high to low frequency): gamma rays, X-rays, ultraviolet
radiation, visible radiation, infrared radiation, microwave radiation, short
radio waves, and long radio waves.
 These categories are somewhat arbitrary in nature and all
electromagnetic radiation share common features.

12.  The key properties of electromagnetic radiation are
intensity, frequency (wavelength), polarization, phase and
angular orbital momentum.
 The first four properties are directly used in PLM.
 Intensity is defined as power over area, for example,
watts/m2. We have an intuitive sense of intensity in
normal life as we may turn on more lights in an area if we
feel it is dimly lit.
 While intensity is of great interest in microscopy, we do
have some control over the property since most
microscopes use artificial illumination with variable power
bulbs.
 Historically, intensity was a major issue in microscopy
since most microscopes used the sun or candles as the
light source.
 Even today, we are interested in increased intensity for low
light level applications like fluorescence microscopy.
 For that reason, some specialized techniques also have
specialized high-intensity light sources.

13.  Optical crystallographic references are scattered and
incomplete and there are relatively few scientists that
are skilled in the necessary techniques.
 I believe that there are a number of good reasons to
attempt a revival of optical crystallography in
pharmaceutics.
 First, it does provide a set of unique values for the
identification of solid-state forms.
 Second, optical crystallography can be quite useful in
the study of thermodynamic form relationships.
 Third, if the optical and X-ray crystallographic
characteristics have been related, then it is possible
to determine which faces and thereby which
functional groups of the chemical are exposed by
comminution processes (Nichols, 1998).
 Fourth, a thorough understanding of optical
crystallography is an excellent tool for the
understanding of crystallo-graphy overall.

14.  Electromagnetic radiation can be considered as a wave phenomenon. As
such, the waves have a frequency (number of waves in a unit measure)
and the inverse – a wavelength. Wavelength is frequently used in optical
microscopy and is defined as the wave distance from peak-to-peak or
from trough-to-trough. These relationships can be mathematically
represented as follows:
 where f is the frequency, u is the velocity of the wave, and l is the
wavelength. For the electromagnetic spectrum,
 Since the velocity of the wave is the speed of light. We often use the
term and concept of wavelength in optical microscopy and commonly
use nanometers as the unit of measure (spectroscopy more commonly
uses wavenumber,
 Various filters of specific wavelengths of light are used with the
microscope and the most common of these are 589 nm (D line), 486 nm
(F line), and 656 nm (C line). The D line (589 nm) is yellow light and is
situated in the middle of the visible spectrum.
 Some microscopists also use monochromators with their microscope to
produce narrow wavelengths of light, although the practice is not as
common today as it once was.

15.  Polarization refers to limitations on the direction of wave
oscillation.
 A polarizer acts like a filter to allow only light oscillating in
one orientation to pass.
 We use polarized light for many applications in optical
microscopy.
 For example, we can determine crystallinity of a sample
using a few crystals and crossed polars. The test is
sensitive, fast, and prone to only a few errors.
 Polarization is one of the more difficult concepts to
describe since there really are not many large scale
analogous phenomena to act as an example.
 As Needham (1958, pg 164) states, “It is much easier to
demonstrate polarized light than to clearly describe what
it is.” I think I could title this entire chapter with that
statement.
 Bloss (1961) presents a cogent and wellillustrated
introduction to polarized light.

16.  Phase refers the time progression of the wave.
 In other words, at t0 the wave may be at a peak, t1 halfway
between peak and trough, t2 at the bottom of the trough, etc.
 In general, we do not use phase information directly in
microscopy but only as the phase relates to other waves through
interference.
 Light waves can either constructively or destructively interfere.
 The waves may add in amplitude, subtract in amplitude, or
cancel each other.
 If the phase of the combining light waves is the same, the waves
will constructively interfere and add amplitudes.
 If the waves are out of phase, then amplitudes will be
diminished or even canceled.
 Phase differences are used in a variety of microscopy techniques
primarily to improve contrast.
 Phase contrast, differential interference contrast, and Hoffman
modulation contrast are just a few of the methods utilizing
phase differences to enhance contrast.
 These latter techniques are more commonly applied to biological
than to physical pharmacy.

17.  Some crystals have the property of double refraction and
incoming light is split into different optical axes (directions
where speed of light is different).
 When this light is recombined, such as in a polarizing light
microscope, we get interference and with PLM a visual
interferogram is formed at the back focal plane of the objective.
 The properties of this interference figure are characteristic of
the material and can be used in understanding the
crystallography and molecular properties of that material.
 The underlying physical principle is alteration of light speed by
interactions of light with different functional groups in the
molecule.
 The properties of the interference figure can also be used to
identify the crystal and even to distinguish among polymorphs,
hydrates, and solvates.
 Double refraction is the phenomenon underlying all of optical
crystallography and it behooves us to understand it well. Simple
refraction occurs when a light beam traverses a refractive index
interface.
 Another important property of light waves is diffraction as the
light interacts with a slit or small opening. In a sense, light bends
around the opening so that it appears that the small slit or hole
is the source of new waves.

18.  It is not necessary to be an expert at optics to intelligently use
the microscope, but it is necessary to know and understand a
few basic concepts.
 There are many excellent texts on optics in the microscope and I
recommend Slayter and Slayter (1992), McCrone et al. (1984),
and Chamot and Mason (1958) along with Needham (1958) for
further study.
 At the heart of optics is the interaction of light with materials
and, of course, for microscopy the material of most interest is
glass in different shapes.
 When visible light irradiates an object, the light can be reflected,
transmitted, or both.
 Reflection and transmission can occur such that the light retains
all of its energy or some energy can be absorbed in the process.
 The rich complexity of the interaction of light with objects, in
particular glass, allows for the versatility and power of modern
microscopes.

19.  The law of reflection states that the angle of reflection is equal to
the angle of incidence for any particular light ray irradiating a
smooth, reflective surface.
 If the surface is irregular and a broad beam of light is used, then
we get diffuse reflection.
 If the surface is polished and we use a narrow beam of light,
then we get specular reflection and this reflected light can be
polarized.
 Fishermen are familiar with this phenomenon and use polarizing
sunglasses to reduce light reflection so as to better see the fish
below the surface of the water.
 It is easy to see that if we make mirrors of different shapes,
particularly concave mirrors, we can use the curved surface to
focus light rays to a point and produce an image.

20.  According to Webster’s Ninth New Collegiate Dictionary (Merriam
Webster 1983), crystallography is defined as the “Science dealing
with the system of forms among crystals, their structure, and
their forms of aggregation.”
 The atoms or molecules of a crystalline solid usually are
oriented in space with a repeating pattern in three dimensions.
 Think of bricks in a wall or even better, of patterned ceramic
tiles. This is the crystalline state.
 In some solids, there is no long-range repeating pattern, though
there may be short range order and this condition is referred to
as the amorphous or glassy state.
 There are also imperfections in most real crystals and this
condition is referred to as disorder.
 Crystallinity and disorder are important topics in physical
pharmacy since the degree of Crystallinity can affect some
important bulk properties of a solid such as the amount of water
it can absorb as a function of temperature and relative humidity.

21.  Surprisingly, there are only a few repeating patterns.
 The patterns can be categorized as follows into six crystal
systems with decreasing degrees of symmetry: cubic, tetragonal,
hexagonal, orthorhombic, monoclinic, and triclinic.
 The trigonal system, which is referenced in older literature, is
now considered part of the hexagonal system.

22.  Optical crystallography is most commonly applied in
mineralogy but is also directly applicable to organic
chemicals.
 Optical crystallography is based on the fact that light
travels with different speeds in different directions in the
crystal.
 The methods of optical crystallography take advantage of
this property.
 Given the range and specificity of optical crystallographic
properties of pharmaceutical compounds and a good
reference database, it is possible to identify single small
crystals even to the point of distinguishing between
similar polymorphs.
 Add in thermal microscopy, IR and Raman microscopy,
and SEM/EDS and we have a powerful identification
scheme, indeed.

23.  Table Description of optical properties of crystals
 Optical property Description
 Color Color of thick sections in brightfield
 Habit, shape Habit for well-shaped crystals, morphology of
particles Aggregation, Crystals or particles mechanically or
chemically fused together
agglomeration Appearance and resistance to breakage
Twining Individual crystals sharing face(s)
Cleavage Description of manner in which crystals break
Surface texture Appearance of crystal, particle surface
Transparency How easily does light pass through particle
Edge angles Angles between crystal faces
Dispersion staining colors Central stop colors in high dispersion R.I.
liquids near refractive index of particles
Pleochroism Color change on stage rotation
Refractive indices Formally, speed of light in vacuum divided by
speed of light in particle. Measured
by comparing particle relief in known
R.I. liquids. Number of principal
indices dependant on crystal
system
R.I. Dispersion Variation of refractive index with wavelength
Interference colors Color of crystal between
crossed polars, depends on
birefringence, crystal thickness, and crystal
orientation. Saturation of color dependant on
order

24.  Anomalous interference colors Different sequence of colors related to crystal
thickness
 Birefringence Difference between high and low refractive indices
Extinction position All crystals and crystal fragments will go dark four
times on rotation of the stage (unless the crystal is
oriented so that the optic axis in parallel to the light
path). Refractive index measurements are generally
made at extinction positions for anisotropic crystals
 Extinction angle The angle of a crystal edges with the positions of the
crossed polars (generally aligned with crosshairs of
the eyepiece) when the crystal is dark. Only
pertinent to crystals in the monoclinic and triclinic
crystal systems
 Dispersion of extinction angles Variation of extinction angle with wavelength
Interference figure. An image of light interference at
the back focal plane of an objective for anisotropic
crystals. Uniaxial interference figures have Maltese
cross appearance or part of the cross depending on
crystal orientation. Biaxial interference figures have
appearance of hyperbola or parts of the hyperbola
depending on crystal orientation and optic axial
angle
 Optic sign Sign of birefringence, for uniaxial crystals if e > w
optic sign if positive, negative if opposite. For
biaxial crystals, if value of b closer to that of g, optic
sign if negative, if closer to a then optic sign is
positive
 Optic axial angle Angle between optic axes, evidenced as angle
between dark regions in interference figure. Is 90º
for uniaxial and orthorhombic crystals, and between
0 and 90º for biaxial crystals (monoclinic and
triclinic)

25.  Dispersion of optic axes Variation of angle of optic axes with
wavelength
 Acute bisectrix Vibration direction isecting acute
angle between optic axes
 Obtuse bisectrix Vibration direction
bisecting obtuse angle
between optic axes
 Optical orientation Relationship between
crystallographic directions and
optical directions. Fixed in
uniaxial crystals, but varied
in biaxial
 Sign of elongation For elongated uniaxial crystals
(and fibers that act like uniaxial
crystals), if the high index is
associated with the length then the sign
is positive, negative otherwise.
Not related to crystal symmetry

27.  This is the simplest type of microscope in terms of both construction and use.
 The stereomicroscope consists of two compound microscopes which are aligned
side by-side at the correct visual angle to provide a true stereoscopic image.
 The long working distance (space between the specimen and objective lens),
upright non reversed image and large field of view make these the instruments of
choice for performing preliminary examinations of evidence as well as
manipulating small particles and fibers to prepare them for more detailed
microscopical or instrumental analyses or comparisons.
 An additional advantage which results from the long working distance and
illumination by reflected light is that specimens rarely require any sample
preparation.
 The specimen is simply placed under the microscope and observed.
 The useful magnification range of stereomicroscopes is typically between 2.5 x
and about 100 x .
 Modern stereomicroscopes incorporate a number of features which increase their
utility and ease of use.
 A choice of illuminators which can provide brightfield and darkfield reflected,
fluorescence and transmitted light permit the microscopist to visualize
microscopic objects and features which might otherwise appear invisible, and thus
escape detection.

28.  Attaching the microscope to a boom stand permits it to be
swung out over large objects such as clothing, piles of debris or
even entire vehicles.
 Both photographic and video cameras can be attached to record
images for inclusion in a report, as a courtroom exhibit or to
display to colleagues.
 Even the least experienced members of the laboratory staff can
use these instruments with very little training

29.  The comparison microscope is used to compare microscopic items side
by side.
 Although the human eye can be very good at discerning minute
differences in color and morphology, the brain has a more difficult time
remembering and processing these subtle differences.
 This problem is overcome by a comparison microscope in which the
images from two microscopes are observed side by side in a single field
of view.
 Reflected light instruments are used by firearms examiners to compare
rifling marks on bullets as well as ejector marks and firing pin
impressions on cartridge cases.
 Tool marks and cut and polished layered paint chips can also be
compared with the same equipment.
 Transmitted light microscopes are used to compare hairs, fibers and
layered paint chips which have been thin-sectioned. Polarizing and
fluorescence equipment may added to a comparison microscope which
is to be used for fiber comparisons to enhance its capabilities.
 Human hair comparisons, particularly the final stages of an
examination, are conducted almost exclusively under a comparison
microscope.

31.  The phase contrast microscope is used primarily in serological and glass
examinations.
 Its principal use in serology is to observe cells in biological fluids or after
reconstitution in aqueous mountants.
 Under these conditions cells exhibit a very small optical path difference with
respect to the medium in which they are immersed.
 Such specimens are referred to as phase objects. The human eye cannot observe
phase differences, but it can discern amplitude (dark and light) differences.
 A phase contrast microscope uses half-silvered rings and disks placed in the
optical system to change these phase differences into amplitude differences which
can then be observed and photographed.
 Spermatozoa, epithelial cells, and other cellular matter can be studied in detail
without staining using this technique.
 One of the principal methods of glass comparison is based on a very accurate
measurement of the refractive indexes of the known and questioned samples.
 The measurement is conducted in a hot stage mounted on a phase contrast
microscope.
 The crushed glass fragment is mounted between a slide and cover-slip in a
specially prepared and characterized silicone oil which is placed in the hot stage.
 As the temperature is raised, the refractive index of the silicone oil decreases
while that of the glass remains essentially constant.

33.  Since even small differences between the refractive indexes of the glass and oil are
easily seen with phase contrast, the true match point (i.e. temperature at which
the silicone oil has the same refractive index as the glass) can be observed with
great precision.
 The refractive index of a glass particle can be measured to 0.00002 using this
technique.
 A commercial instrument in which the phase contrast microscope, hot stage and
camera are all connected to a computer makes these measurements automatically
and objectively.
 Fluorescence microscopy is based on the property of certain substances to emit
light of a longer wavelength after they have been irradiated with light of a shorter
wavelength.
 This emitted light is called fluorescence and differs from luminescence in that the
emission of light stops after the exciting radiation is switched off.
 The fluorescence may originate from fluorescent ‘tags’ attached to proteins or
other compounds which cause the substance they react with to fluoresce
 The comparison microscope is used to compare microscopic items side by side.
Although the human eye can be very good at discerning minute differences in
color and morphology, the brain has a more difficult time remembering and
processing these subtle differences.
 This problem is overcome by a comparison microscope in which the images from
two microscopes are observed side by side in a single field of view.
 Reflected light instruments are used by firearms examiners to compare rifling
marks on bullets as well as ejector marks and firing pin impressions on cartridge
cases.

34.  Tool marks and cut and polished layered paint chips can also be
compared with the same equipment.
 Transmitted light microscopes are used to compare hairs, fibers and
layered paint chips which have been thin-sectioned.
 Polarizing and fluorescence equipment may added to a comparison
microscope which is to be used for fiber comparisons to enhance its
capabilities.
 Human hair comparisons, particularly the final stages of an examination,
are conducted almost exclusively under a comparison microscope. after
the nonreacting remainder of the reagent is washed away, or it may
originate from autofluorescence.
 The first technique is the basis for detecting antigen-antibody reactions
which occur on a cellular level and has been applied to a limited extent
in forensic serology.
 Autofluorescence may originate from either organic or inorganic
compounds or elements.
 When it occurs, autofluorescence is a useful comparison characteristic.
 It may originate from organic dyes or optical brighteners on fibers; it
may be observed in layers of paint in cross-section where it originates
from organic pigments or inorganic extenders and may be observed on
certain varieties of mineral grains and be absent from others
 A modern fluorescence microscope is equipped with a vertical
illuminator which directs the light from a mercury burner through a
series of lenses and filters designed to focus the light on the specimen
and select a narrow or wide range of wavelengths to excite fluorescence
in the specimen.

36.  Since the intensity of the fluorescence from a specimen does not depend on
absorption and the image is formed with the emitted fluorescent light rays,
fluorescence images are bright and well resolved.
 These images can be recorded and make excellent exhibits for use in
reports and courtroom presentations
 The hot stage microscope permits the microscopist to observe the behavior
of specimens as they are exposed to temperatures from ambient up to
approximately 350°C.
 Melting temperatures can be used to help in the identification of unknown
substances and as an aid in certain types of comparisons; particularly those
involving thermoplastic polymers.
 For example, infrared microspectroscopy is of only limited use in
distinguishing nylon fibers.
 It can be used to determine if fibers have been spun from nylon 6 or nylon
6,6 polymer.
 Much finer distinctions can be made by comparing melting points of nylon
fibers since these are a function not only of the type of polymer from which
the fiber was spun, but also the average molecular weight, crystallinity,
presence of additives, etc.
 Although the contributions from each of these factors cannot be individually
assessed from a melting point determination alone, the actual melting points
of two fibers result from all of these factors and thus form a useful point of
comparison or discrimination.
 Although other instrumental methods of analysis have largely superseded
hot stage microscopy as a tool for the identification of unknown compounds,

37.  Electron microscopes make use of electrons rather than photons to form
their image. The transmission electron microscope (TEM) was developed
first, followed some years later by the scanning electron microscope
(SEM).
 Transmission instruments are generally more difficult to use and
require more painstaking sample preparation than scanning microscopes
and thus have found very few applications in forensic science.
 Specimens for TEM must be extremely thin to permit penetration by the
electron beam.
 The image in an SEM is formed from collected secondary or
backscattered electrons emitted from (and just beneath) the surface of
the sample and not by transmitted electrons as in the TEM.
 Since the SEM only looks at the surface of a specimen, sample
preparation is often much simpler and frequently consists simply of
placing the specimen on a piece of conductive carbon tape.
 It may be necessary to vacuum deposit a layer of carbon or gold over
nonconductive specimens to make them conductive, although the new
‘environmental SEMs’ can image nonconductive samples in a low
vacuum. SEMs are now in use in many forensic laboratories around the
world.
 Most of these microscopes are equipped with energy dispersive X-
ray spectrometers for elemental analysis.

38.  X-ray spectrometers collect the X-rays which are produced along with the
secondary and backscattered electrons when a specimen is bombarded in a
vacuum with electrons.
 These X-rays are collected and then sorted in a multichannel analyzer
according to their energy which is directly related to atomic number.
 Both qualitative and quantitative analyses can be performed on microscopic
specimens from boron all the way up in the periodic table.
 The detection limit for each element varies, but typical limits of detection for
most elements, excluding some of the light elements, is about 0.1%. One of
the principal uses of analytical SEMs in forensic science laboratories is the
detection and analysis of gun shot residue (GSR) particles.
 Conductive sticky tape, attached to the back of a sample stub, is pressed
over a suspect’s hands to collect any residue which might be present.
 The stub is placed in the microscope and searched, either manually or
automatically, for particles with a spherical morphology which contain lead,
antimony and barium.
 The combination of the spherical morphology with this elemental
composition provides better proof of the presence of GSR than an elemental
analysis alone.
 Other types of microscopic evidence which can be examined in the SEM
include items as diverse as pollen grains, diatoms, paint, glass, inorganic
explosives and general unknowns.
 The combined abilities of the SEM to resolve fine structures and provide the
elemental composition of these small particles is a tremendous aid in the
examination of many small items of trace evidence