Forensic Trace Evidence: Microscopy, Analysis, and Legal Standards

Trace Evidence

Microscopic material recovered as evidence that is used to help solve criminal cases.

Trace Evidence Analyst / Forensic Scientist

Concerned with the characterization, identification, and comparison of microscopic materials in criminal cases.

Primary Transfer (Direct Transfer)

The direct transfer of evidence from the source to a person or object.

Secondary Transfer

The passive or secondary transfer of evidence.

1:1 Taping

The epitome of localized evidence collection where the area covered by one piece of tape represents the same area of the surface taped.

Bulk Evidence

Bulk material that may have contributed to the trace evidence must be collected as standards for comparison.

Microscopy (Role)

Allows a magnified view of the evidence for further identification/separation.

Polymorphs

Substances that have the same chemical composition but can crystallize with different internal structures.

Polarized Light Microscopy (PLM)

A microscopy technique that serves as the basis for the identification of synthetic fibers and for mineralogical examinations.

American Board of Criminalists (ABC)

An organization in the US that offers certification of trace evidence examiners.

Council for Registration of Forensic Practitioners (CRFP)

Established in the UK with a team of expert assessors who process examiners' applications for certification in various subject areas.

Real Evidence

Evidence that is directly related to and collected from the crime scene.

Demonstrative Evidence

Evidence not directly collected from the crime scene, but developed at later stages to assist and understand the importance of real evidence.

Chemical Forensic Samples

A classification including drugs, toxins, fire and arson samples, explosives, and petroleum products.

Body Fluids (Biological)

Biological forensic samples including blood, semen, saliva, vomit, milk, vaginal secretion, sweat, tears, and urine.

Non-body Fluids (Biological)

Biological forensic samples mainly including teeth, nails, feces, bone, hair, and tissue.

Botanical Forensic Samples (Preservation)

Samples collected and preserved by pressing and drying in natural conditions to maintain physical features.

Quality Assurance (QA)

A set of planned or systematic protocols developed to ensure that a product or service is of utmost quality.

Quality Control (QC)

Refers to the day-to-day operational protocols and activities designed to ensure the fulfillment of the quality requirements of a product or service.

ISO 17025

A standard defining the general responsibilities and requirements to check the competence of testing and calibration laboratories.

ISO 17020

A standard responsible for stating the different requirements of the organizations that undertake the inspection of forensic laboratories.

ISO 15189

A standard responsible for analyzing the quality assurance and management of medical laboratories.

Standard Development Organizations (SDOs)

A group of bodies responsible for quality management in various laboratories and organizations.

Level 2 Organizations (SDOs)

Organizations responsible for selecting, adapting, and interpreting Level 1 standards according to the requirements of the laboratory (e.g., the Internal Laboratory Accreditation Guide by ILAC).

Standard Operating Procedures (SOPs)

Extremely detailed protocols for carrying out policies or protocols and operating equipment, which staff must adhere to stringently.

Chain of Custody (QA/QC)

A highly imperative documentation process for every sample obtained from a crime scene, including the amount, type, date, and origin of collection.

Standard Reference Materials (SRMs) / Certified Reference Materials (CRMs)

Materials that are routinely analyzed for Quality Control purposes in forensic science.

Blind Drug Samples

Control samples that are unknown to the person doing the analysis; they are considered the best control samples for efficient QC in drug analysis.

Ethics

A branch of philosophy derived to oversee the morality of a human being, designed to protect the rights and requirements of a person.

Deposition

Testimony given by taking an oath but not in front of a judge or in court.

Discovery

A legal process in which both sides seek to collect data about the evidence the opposite side possesses.

Reciprocal Discovery

The term used when both parties (prosecution and defense) can legally inspect each other's evidence.

Subpoena Duces Tecum

A specific subpoena for forensic scientists requiring the witness to submit both their testimony and all relevant evidence or documents in their possession to the court.

Lay Witness (Nonexpert Witness)

A witness who is legally only allowed to report sensory observations (sight, sound, smell, etc.) and is not allowed to give any kind of opinion.

Expert Witness

Generally the forensic scientist who performed the analysis; allowed to give opinions and conclusions based on their expertise and findings.

Chain of Custody (Legal)

The proper and legal channel of labels and sponsors used to maintain the authenticity of evidence from the time of seizing until submission in court.

Competence (of Evidence)

A requirement for evidence admissibility; the evidence must not be prejudiced, time wasting, unreliable, or presented through improper procedure.

Stereomicroscope

Typically used to image a sample with a lower magnification. It uses reflected light rather than transmitted light, allowing it to image opaque or thick samples.

Bright-Field Microscope (BFM)

One of the simplest compound microscopes, widely used for the preliminary analysis of forensic evidence, but limited by low contrast and resolution.

Numerical Aperture (NA)

A crucial factor in microscopy that is the ability of the lens to clearly differentiate the positions of two closely situated points. The greater the NA, the greater the resolving power of the lens.

Polarized Light

Light in which the vectors of light oscillate on only one axis.

Refractive Index (RI)

Represents the speed change of light as it moves through different mediums.

Phase Contrast Microscope (PCM)

Exploits the principle that light speed changes in different mediums (RI) to create contrast, resulting in dark and well-defined structures against a bright background.

Stoke's Shift (in Fluorescence)

The phenomenon where the energy released (fluorescence) when an electron falls back to the ground state is less than the excitation energy absorbed, thus releasing a wave that has less energy and is longer.

Confocal Microscope

A type of optical microscope that increases the resolution and contrast of an image by focusing on it through a pinhole, enabling the construction of a three-dimensional (3D) view.

Birefringence

A phenomenon typically occurring in anisotropic materials where the refractive index changes based on the polarization and direction of light propagation, causing the polarized light to split into two rays.

Scanning Electron Microscope (SEM) / Transmission Electron Microscope (TEM)

Types of electron microscopes capable of visualizing an object in the range of pico- to nanometers, since light microscope resolution does not exceed 0.1 μm.

Energy Dispersive X-ray Spectroscopy (EDX or EDS)

A technique that can be combined with electron microscopes to analyze the elemental composition of the material.

Secondary Electrons (SEM)

Electrons that escape from the material once the primary electron has hit the sample; they provide information about the sample topography and form.

Backscattered Electrons (SEM)

Electrons that reflect from the specimen; they give an idea about the multiple phases existing in the composition of the material.

Elastic Scattering

Electron scattering where the energy is conserved when the electron hits the material; allows the imaging of crystalline structures with greater resolution.

Inelastic Scattering

Electron scattering where there is a loss in energy upon impact; provides information about the thickness, volume, and transparency of the material.

UV-Visible Spectroscopy (UV-Vis)

A basic analytical technique based on the absorption of radiation in the UV-visible region, causing electronic transitions (electrons moving from a ground state to an excited state).

Beer-Lambert Law

An expression (A = εlc) used in UV-Vis spectroscopy to correlate the light absorbed (A) to the sample's concentration (c) and absorptivity (ε).

Lambert Law

States that absorbance (A) is the negative logarithm of the fraction of transmitted light (T).

Monochromator

Components (gratings, mirrors, and filters) used in UV-Vis spectroscopy that work together to produce monochromatic light (a single wavelength at a time).

Fluorescence Spectroscopy

Based on the emission of energy (fluorescence) when excited electrons travel from the excited state back to the ground state.

Fluorophore

A component required in a molecule for fluorescence spectroscopy; responsible for emitting energies with wavelengths longer than the excitation energy during relaxation.

Photobleaching

The gradual loss of a sample's fluorescence if exposed to a very strong radiation source.

Infrared (IR) Spectroscopy

Based on the absorption of IR light, which causes the sample molecules to vibrate (stretching or bending of bonds). The molecule must possess an electric dipole moment to be IR active.

Heteronuclear Molecules

Molecules consisting of two different atoms where an electric dipole is created, making them capable of vibrating upon IR absorption.

Homonuclear Molecules

Molecules that have an electric dipole of zero and therefore are said to be IR inactive (e.g., two or more of the same element).

Fingerprint Region

The crucial part of the mid-IR region (400-1400 $ ext{cm}^{-1}$) that is responsible for the unique characteristic signal of every molecule.

Overtone Bands (NIR)

Bands that arise when there are multiple fundamental absorption frequencies.

Combination Bands (NIR)

Bands formed when two fundamental bands absorb energy simultaneously, causing peaks to overlap.

Fourier Transform IR (FTIR)

An IR technique that uses a Michelson interferometer to generate an interferogram (a signal produced due to the change in path length between two different beams) which is then converted into a spectrum via Fourier transform.

Attenuated Total Reflectance (ATR)

An FTIR sampling technique in which the IR beam is sent directly to a dense crystal, and the sample is analyzed without extensive sample preparation by measuring the multiple reflectance that occurs.

Raman Spectroscopy (RS)

Works on the basis of Raman scattering, a type of inelastic scattering that occurs when photons hit a molecule, resulting in a slight energy difference from the initial photon's energy.

Stokes Shift (Raman)

The phenomenon where the scattered energy has slightly less energy than the incident light; this produces a stronger signal and is often the preferred shift during analysis.

Anti-Stokes Shift (Raman)

The phenomenon where the scattered light tends to have more energy than the incident light; it is significantly weaker than the Stokes shift but preferred when interference such as fluorescence is present.

Gas Chromatography (GC)

A powerful analytical tool used for the analysis of various forensic evidence that can be made volatile.

Stationary Phase (GC)

The solid coating on the inside of the GC column to which analytes briefly adsorb.

Mobile Phase (GC)

The carrier gas (e.g., helium or hydrogen) that is forced through the column and carries the samples from the inlet to the detector.

Average Linear Velocity (GC)

A representative of the velocity of the gas within the column according to the pressure inside it; more useful than volumetric flow because it is independent of the volume of the column.

Retention Time (GC)

The characteristic amount of time a sample spends retained on the stationary phase and, therefore, in the column.

Packed Column (GC)

A column consisting of small particles typically of diatomaceous earths that are usually coated with various organosilanes to enhance the adsorption capacity of the stationary phase.

Capillary Column (GC)

A simple column with extremely small diameters that does not contain any packing, offering high separation efficiency.

Thermal Conductivity Detector (TCD)

A GC detector that measures the difference in the amount of power required to maintain the temperature of two filaments—one exposed only to the carrier gas, and one exposed to the carrier gas with the sample.

Flame Ionization Detector (FID)

A GC detector highly suitable for detecting low concentrations of analytes by detecting the ions formed when an analyte is burned in a hydrogen flame.

Electron Capture Detector (ECD)

A GC detector that works by capturing electron-absorbing compounds such as halogens present in the carrier gas.

High-Performance Liquid Chromatography (HPLC)

An advanced liquid chromatography technique that employs high pressure to push the mobile phase through a densely packed stationary phase. It is suitable for analyzing compounds that are nonvolatile, have high molecular weight, and are thermally sensitive.

Forensic Toxicology

The division of forensic science that helps in the legal or medical examination of drug abuse, poisoning, or death due to a drug overdose or the consumption of poison.

Retention Time (HPLC)

The time duration for which a component is present inside the column before elution.

Reverse Phase (HPLC)

A separation mode where the stationary phase is nonpolar while the mobile phase is polar; this technique is more common for hydrophilic drug analysis.

Normal Phase (HPLC)

A separation mode where the mobile phase is nonpolar in nature while the stationary phase is polar.

Ion Exchange (HPLC)

A separation mode where polar compounds and ions are separated on the basis of their charge, typically using a resin in the stationary phase.

Chiral Chromatography (HPLC)

A technique used to separate stereoisomers, specifically enantiomers (stereoisomers with nonsuperimposable mirror images).

Isocratic Elution (HPLC)

Elution where the composition of the mobile phase remains the same throughout the process; suitable only for simple separations.

Gradient Elution (HPLC)

Elution where the composition of the mobile phase keeps on changing during the separation process, resulting in sharper and narrower peaks and assisting in complex separations.

Guard Column (HPLC)

A small column, fitted just before the analytical column, whose purpose is to protect the analytical column from any kind of contamination from foreign particles.

Analytical Column (HPLC)

Considered the heart of the HPLC instrument; typically made of stainless steel and containing packing material (e.g., silica).

Mass Spectrometry (MS)

A powerful technique that targets the mass-to-charge ratio (m/z) of a molecule for its highly sensitive and accurate determination, providing rich elemental information.

Molecular Ion ($\text{M}^{+}$)

The positively charged parent ion of a molecule generated during ionization. Due to its high instability, it further breaks into a single or multiple daughter ions.

Base Peak

The molecular ion ($\text{M}^{+}$) peak with the highest intensity, which corresponds directly to the molecular weight of the compound.

Electron Ionization (EI)

An ionization source where high-energy electrons collide with gas-phase samples, knocking off an electron to create the $\text{M}^{+}$ ion. The high energy can also cause overfragmentation ('shatters') of the molecule.

Chemical Ionization (CI) / Soft Ionization

An ionization technique for liquid samples where the fragmentation is less extensive, due to which a simpler and clearer spectrum is formed. Processes include proton transfer, charged species transfer, or adduct formation.

Secondary Ion Mass Spectrometry (SIMS)

A technique preferably used for the analysis of solid samples, detecting secondary ions emitted when high-intensity electrons hit a surface.

Matrix-Assisted Laser Desorption Ionization (MALDI)

A technique where the sample is dissolved in a matrix, dried, and ionized using a laser. Due to the isolation of the sample molecules by the matrix, there is a very minimal chance of formation of sample clusters, meaning the sensitivity of the analysis is very high.

Mass Analyzers

Components used to separate the ions produced during the ionization stage based on their mass-to-charge ratio (m/z).

Quadrupole Analyzers

Mass analyzers consisting of four parallel rods that separate ions based on the stability of the trajectories of the ions, controlled by the fluctuation of electric potentials.