FORENSIC MICROSCOPY

Earliest Microscopes

A. Antonie van Leeuwenhoek (1632-1723)

• 1673- Deft, Holland, worked as a draper (fabric merchannt); he is also known
to have worked as a surveyor, a wine assayer, and as a minor city official.
Leeuwenhoek is incorrectly called “the inventor of the microscope”
• Created ‘simple’ microscope that could magnify to about 275x and
published drawings of microorganisms in 1683
• Could reach magnifications of over 200x with simple ground lenses- however
compound microscopes were mostly of poor quality and could only magnify
up to 20-30 times/ Hooke claimed they were too difficult to use- his eyesight
was poor.
• Discovered bacteria, free-living and parasitic microscopic protists, sperm
cells, blood cells, microscopic nematodes.
• In 1673, Leeuwenhoek began writing letters to the Royal Society of London-
published in Philosophical Transactions of the Royal Society.
• In 1680, he was elected a full member of the Ro yal Society, joining Robert
Hooke, Henry Oldenberg, Robert Boyle, Christopher Wren.

Secondary Microscopes
B. George Adams Sr.
• Made many microscopes from about 1740-1772 but he was predominantly
just a good manufacturer not inventor (it is believed he was a copier)
• Simple microscopes could attain around 2-micron resolution, while the best
compound microscopes were limited to around 5 microns because of
chromatic aberration.

C. Chester More Hall

• During the 1730s, a barrister named Chester More Hall observed that a flint
glass (newly made glass) dispersed colors much more than “crown glass”
(older glass). He designed a system that used a concave lens next to a
convex lens whicih could realign all the colors. This was the first achromatic
lens. George Bass was the lens-maker that actually made the lenses, but he
did not divulge the secret until over 20 years later to John Dolland who
copied the idea in 1759 and patented the achromatic lens.

D. Giovanni Battista Amici

• In 1827, he built high quality microscopes and introduced the first matched
achromatic microscope in 1827. he had previously (1813 designed “reflecting
microscopes” using curved mirrors rather than lenses. He recognized the
importance of coverslip thickness and developed the concept of “water
immersion”
Some Definitions

• Absorption
➢ When light passes through an object the intensity is reduced
depending upon the color absorbed. Thus, the selective absorption of
white light produces colored light.
• Refraction
➢ Direction change of a ray of light passing from
one transparent medium to another with different
optical density. A ray from less to denser medium
is bent perpendicular to the surface, with greater
deviation for shorter wavelengths.

• Diffraction
➢ Light rays bend around edges- new wave fronts
are generated at sharp edges – the smaller the
aperture the lower definition
 • Dispersion
➢ Separation of light into its constituent wavelengths when entering a
transparent medium- the change of refractive index with wavelength,
such as the spectrum produced by a prism or a rainbow

• Magnification
➢ An object can be focused generally no closer than 250 mm from the
eye (depending upon how old you are)
➢ This is considered to be the normal viewing distance for 1x
magnification
➢ Young people may be able to focus as close as 125 mm so they can
magnify as much as 2x because the image covers a larger part of the
retina- that is it is “magnified” at the place where the image is formed.

The applications of microscopy in the forensic sciences are almost limitless.

This is due in large measure to the ability of microscopes to detect, solve,

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 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 was 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 microscopical
examination begins is often essential. Typical examples of sample preparation might
include: mounting a crystal in index of refraction oils to determine its optical
properties, reconstituting a blood crust particle and staining it for leukocytes and
other cells, preparing a cross-section of a fiber or mounting a specimen in a
nonfluorescent mounting medium to observe its autofluorescence.
As a general rule, the type of specimen, the information one wishes to obtain
from it and the type of microscope chosen for the task will determine if sample
preparation is required and the type of processing required (Epi-Flour Microscopes).
Forensic Microscope is mainly used by forensic science experts to help them with
their crime investigations. It has the ability to identify and compare the proofs
collected to justify that a victim, suspect or the place of crime had contact on each
other, it is valuable tool for investigators I tracing pieces of evidence that are often
small and cannot be seen with the human eye.

This type of microscope also work on forensic ballistics which is the study of
criminal law and answering technological questions arising while the investigation of
crime is in process. This study is on the objects like handguns, ammunition, bullet
holes and traces of a close shot, thermal effect of powder gases.

Nowadays, forensic microscopy become an exciting field for many forensic

science experts, investigators, the law, and the criminology students. Nevertheless,
the most important purpose of forensic microscopes is to help investigators resolve
crime cases and contribute to the accuracy of judgments (Welcome of Forensic
Microscope, 20130.)

Today, forensic scientists can choose from a variety of techniques to study this
evidence, but perhaps the most important technique has been forensic microscopy.
Forensic microscopy encompasses the identification and classification of a wide
range of materials and substances: impressions such as fingerprints and foot prints,
fractured fragments such as broken tools and torn paper, trace evidence such as
hairs and fibers, genetic markers, bullets, and handwriting.

The tools employed in forensic microscopy vary from the basic low power
hand magnifiers to high power electron microscopes:
a. Binocular (stereo) microscopes are commonly used in document
examination, as in trying to determine whether one pen stroke passes over
another
b. Scanning electron microscopes are used for morphological and elemental
analysis, and to identify gunshot residue (GSR)
c. Transmission electron microscopes are used for pathogen analysis and for the
examination of paint pigments
d. Infrared or IR microscopes are used in drug identification, while
e. Phase contrast microscopes are used to characterize materials such as glass
and biological fluids.

Types of Microscopes Used in Forensic Sciences

A variety of microscopes are used in any modern science laboratory. Most of

these are:
a. Light microscopes which use photons to form images
b. 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

A. Stereomicroscope
• This is the simplest type of microscope
in terms of both construction and use
• 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 instruments of choice for
performing preliminary examination
of evidence as well as manipulating
small particles and fibers to prepare them for more detailed microscopical or
instrumental analyses of comparisons.
• Specimens rarely require any sample preparation. The specimen placed
under the microscope and observed
• The useful magnification range of stereomicroscopes is typically between 2.5x
and about 100x
• Modern stereomicroscopes have choice of illuminations which can provide
bright field and dark field, fluorescence and transmitted light permit the
microscopist to visualize microscopic objects and features which might
otherwise appear invisible, and thus escape detection.
• 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.

B. Compound Microscope
• Compound microscopes
represent a significant step up
in magnification, resolution,
and difficulty of use from the
stereomicroscope.
• Magnifications range from 2.5x
to about 1300x with a
corresponding increase in
resolving power
• Most observations transmitted
light which places limitations
 on the specimens which are to be studied
• Reflected light instruments, used to study bullets and tool marks, have found
limited use in forensic labs and are generally confined to the examination of
metals that have been prepared by grinding and polishing
• A variety of compound microscopes are available to the forensic
microscopist and their selection will depend on the types of evidence to be
studied. These include:
o Standard bright field;
o Phase contrast, comparison, hot stage, fluorescence and polarizing
microscopes.
o Bright field microscopy is used to observe and study the morphology of
microscopic specimens. In the forensic lab these can include a range
of materials almost numerous to list.

C. Polarizing microscope
• Most useful and versatile instrument in the
hands of a trained and experienced forensic
microscopist.
• Allows transparent solids to be examined in
plane polarized light (to isolate unique
vibration directions in a crystal or crystalline
polymer), between crossed polars.

Table 1. Types of Evidence through Morphology

Type of Sample preparation Identification features observed
Evidence required
Hairs Preparation on glass slides Color. Overall shape and length.
in mounting medium. Scale Scale patterns. Cross-sectional shape
impressions. Cross-sections. and structural features. Pigmentation
Clearing medulla distribution. Cellular structure of
medulla
Vegetable Scraping to look for General color and texture. Shape of
fibers epidermal and vascular any crystals recovered. Appearance
tissue and crystals, of epidermis and other cells. Features
Maceration into fiber of fiber ultimates: length, structure of
ultimates. Micro chemical lumen, cell walls and pits. Comparison
testing for lignin content to reference slides
 Wood Preparation of 3 sections: Recognition of arrangements of
transverse, radial and tracheids or fibers and vessel
tangential. Mounting in elements. The arrangement of the
glycerin alcohol or cells in all sections. Comparison to
permanent medium and reference slides and illustrations
staining. Even slivers and
sawdust can be sectioned
Pollen Acetolysis (boiling in mixture Shape. Type and arrangement of
of acetic anhydride and apertures: pores and/or furrows.
sulfuric acid) to remove Structure (e.g. collumellae) and
cytoplasm. Mounting in sculpting (e.g. echinate) of the exine.
glycerin, glycerin jelly or Comparison to reference slides and
silicon oil atlas figures.
Diatoms Boiling in strong acids to Shape of girdle and valve views, sixe,
destroy organic matter. arrangement of pores, fine structure
Mounting in high refractive of the central raphid in the center of
index medium the diatom
Blood crust Reconstitute in reagent Recognition of leukocytes and other
such as toluidine blue cells which may give clues as to other
tissues present

In addition to the microscope itself, a set of calibrated index of refraction oils are
required to perform optical crystallographic measurements. Table 2 summarizes the
determinative methods used along with some typical applications.

Table 2. Optical Properties and their Determination with the Polarizing Microscope
Optical Measurement Determinative Value
Property
Refractive Immersion Method. Specimen Identification of unknown
Index placed in index of refraction isotropic crystals. Comparison of
oils until it disappears. glass particles.
Refractive Orientation of principal Crystallographic identification of
indices vibration direction with chemical crystals and minerals.
polarizer of microscope.
Matching of two (uniaxial Identification and comparison of
crystals) or three (biaxial artificial fibers.
crystals) with index of refraction
oils.
Birefringence Numerical difference between Identification of crystalline and
two principal refractive indices. semi-crystalline materials.
By subtracting larger refractive Comparison of certain artificial
index from smaller or by fibers.
measurement of retardation
 with a compensator and
measurement of thickness.
Optic Sign Convention based on relative Aid in the identification of
magnitudes of refractive crystals, minerals, or fibers. For
indices. By comparison of artificial fibers, the more easily
values of refractive indices or determined sign of elongation is
be means of compensators. the same as the optic sign.
Interference Viewed in convergent light Aid in determining the optical
Figure between crossed polars with character of crystals as uniaxial
the objective of high numerical or biaxial. Provides optical
aperture using a Bertrand lens orientation of crystals without
or pinhole. diagnostic external morphology.
Pleochroism Rotation of the crystal, particle, Diagnostic aid in the
or fiber between extinction identification of heavy minerals
positions with only one polarizer and their varieties in soil
inserted in the optical path. minerology. Comparison aid in
the examination of colored
artificial fibers.

D. Phase Contrast Microscope

• 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.
• Uses half-silvered rings and disks placed in the optical system to change
phase differences into amplitude differences which can then be observed
and photographed.
• Spermatozoa, epithelial cells, and other cellular matters can be studied in
detail without staining using this technique.
• The measurement is conducted in a hot stage mounted on a phase contrast
microscope.
E. Fluorescence Microscope
• Based on the property of certain substances to emit light of a longer
wavelength after they have been irradiated with light of a shorter length.
• 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
after the non-reacting remainder of the reagent is washed away, or it may
originate from auto-fluorescence.
• 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.

F. Electron Microscope
• 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 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 of a layer of carbon or gold over
non-conductive specimens to make them conductive, although the new
‘environmental SEMs’ can image non-conductive samples in a low vacuum.
• SEMs are now used in many forensic laboratories around the world. Most of
these microscopes are equipped with energy dispersive X-ray spectrometers
for elemental analysis. X-ray spectrometers collect X-rays which are produced
along with the secondary and backscattered electrons when a specimen is
bombarded in a vacuum with electrons.
• One of the principal uses of analytical SEMs in forensic laboratories is the
detection and analysis of gunshot residue (GSR) particles.

Transmission Electron Microscope

References:
Epi-flour stereomicroscope. (2005). Retrieved from http://what-when-
how.com/forensic-sciences/microscopy/

Eye on forensic microscopy. (2005). Retrieved from

http://www.rdmag.com/articles/2005/eye-forensic-microscopy

Welcome to forensic microscope. (2013). Retrieved from

http://www.forensicmicroscope.com/

Michelle, M. (2007). Uses of forensic microscopes. Retrieved from

https://sciencing.com/uses-microscopes-forensic-science-5523339.html

***Compiled by: Dr. Jun C. Corpuz