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Speaker Abstracts

This is a partial list of Inter/Micro 2019 presentations that will be held on Monday-Wednesday, June 10-12, 2019 in the McCrone Lecture Room.

Abstracts and speakers are subject to change; check this page regularly for additions and updates.

Improved Quantitative Assessment of Carbon Black Dispersion in Polyolefins by Light Microscopy
John R. Reffner and Day-Chyuan Lee — Dow Chemical Company
Carbon black dispersion in polyolefin-based compounds is often assessed using visible light microscopy, and standard test methods such as ASTM D5596-03, ISO 18533, and ISO 11346 have long been established for the quantification. These methods are based on visual assessments of a very limited sample area. In this talk, I will discuss some limitations of these test methods and present a procedure using stage automation/image collection and image analysis. Stage automation allows a large number of images to be collected without operator bias. Image analysis provides quantitative information on both sample thickness and the size of undispersed carbon black domains. The combination provides a rapid and more statistically significant assessment with significantly more sensitivity to low levels of poor dispersion.

“The Particle was Identified as a Cellulose Fiber”
Skip Palenik — Microtrace LLC
In our laboratory, we are frequently asked to identify fibrous particles. New microscopists at Microtrace learn early in their careers that merely identifying an unknown particle as a cellulosic fiber (not a cellulose fiber) usually results in a gross under estimation of the information that such a particle can provide about itself, its history, and its origin.

This presentation will explain and demonstrate some of the characteristics that cellulosic particles can be coaxed into revealing about themselves by the well-prepared observer who has been trained to appreciate the telltale features and attributes, many of which are not obvious even under the microscope, by someone trained to look for them and possessing that most important knowledge of what to look for.

Nanoparticles as Trace Evidence
Kelly Brinsko Beckert, Skip Palenik, and Christopher S. Palenik — Microtrace, LLC
Sub-micron and nanoparticles comprise a new subcategory of trace evidence that is often easily overlooked and thus underutilized in forensic science. Despite the fact that these sub-microscopic particles are nearly ubiquitous in the environment and are found in a number of widely available and commonly used consumer products, from cosmetics to paint and food, they are rarely exploited in casework. This may be due in part to a general lack of awareness regarding the existence of these particles, as well as the fact that methods for their detection, isolation, and analysis are rarely published in the context of forensic science. A protocol based on a published soil separation procedure has been developed, which allows forensic laboratories to utilize familiar equipment and instrumentation for the isolation and analysis of nanoparticles as trace evidence. Special attention is given to background contamination and its larger implications on the interpretation of results. This research demonstrates the efficacy of this technique and shows how nanoparticles or collections of nanoparticles may be used to help characterize soil, dust, or other unknown residues for identifications, comparisons, or the development of investigative leads.

A recent case study illustrates the value of such evidence, examples of some of the techniques that can be used to analyze them, the necessity for caution in interpretation, and the precautions that must be taken and considered in any such investigation.

Tremolite Asbestos from the “Saltworks” Mine, Inyo County, California
Eric J. Chatfield — Chatfield Technical Consulting Limited
Five samples of tremolite asbestos were received over a 39-year period. The available evidence leads to a belief that all five of these tremolite asbestos samples originated from the same mine in Inyo County, CA. The morphological properties of these five samples have been compared by polarized light microscopy (PLM) and transmission electron microscopy (TEM).

Fiber width is known to be a particularly important factor that is associated with carcinogenic activity. However, at the magnification of approximately 10,000x, used for counting of fibers longer than 5 um, the 1 mm increments on the fluorescent screen of a TEM correspond to width increments of 0.1 um. For fiber widths lower than 0.5 um, 0.1 um increments of width do not provide sufficiently accurate measurements. Accordingly, a new fiber counting protocol has been implemented in this work, in which fiber widths below approximately 0.5 um are measured at a magnification of approximately 60,000x.

The fiber widths of two historical samples were found to be significantly thicker than those in samples collected more recently. The fiber size distributions of the three recently collected samples were found to be closely similar and with fiber widths close to those of the Korean tremolite asbestos used in the animal experiments reported in 1991 by Davis, Addison, McIntosh, Miller, and Niven.

How Low Can You Go? Determining the Smallest Steel Fragment for Quantitative Analysis
Peter D. Zoon — Netherlands Forensic Institute
At the Netherlands Forensic Institute (NFI) microtrace analysis of (metal) fragments embedded in bone is a routine part of examinations. These examinations are typically performed with scanning electron microscopy coupled with energy-dispersive spectroscopy (SEM-EDS) analysis to determine the elemental composition of the fragments. Most of the encountered traces are (stainless) steel fragments. As previously discussed, obtaining reliable quantitative elemental compositions with SEM-EDS is not trivial under best of circumstances. To increase the evidentiary value of SEM-EDS analyses of small metal fragments embedded in bone, the quantitative elemental composition of small metal fragments was determined with laser ablation inductively coupled mass spectrometry (LA-ICPMS).

Small samples cut from knife blades showed promising results. These samples are however an order of magnitude larger than the traces typically encountered in bone. A method for generating small fragments without contamination was devised, and the small fragments were analyzed and the obtained compositions have been compared to the composition of the blades.

The smallest fragments that could be reliably analyzed are approximately 50 × 50 um in diameter. The thickness of the fragments was not determined, but it seems reasonable that this should be approximately the same. This lower limit is not governed by the analytical measurement technique, but rather by the nature of the samples themselves.

To conclude this presentation, a case example will be presented in which the above-mentioned analysis was used to determine if metal fragment in the skull bone of a victim could originate from a knife handle.

Sources of GSR Particles: One that Shouldn’t and One that Wasn’t
Martin Janssen — Netherlands Forensic Institute
Gunshot residue (GSR) can play an important role in shooting-incident investigations. For GSR-evidence to be used properly in court, information about the potential sources of the observed particles is critical. In this presentation, two sources are discussed. First a procedure that was in place at the Netherlands Forensic Institute to minimize the transfer of GSR and GSR-like particles from one object to another is shown to be a potential source of GSR-like particles. Secondly, a case in which the defendant successfully claimed a coffee shop to be source of GSR-particles is discussed.

One widely used procedure to prevent GSR-contamination in the lab is to cover the lab bench with fresh and unused paper to create a ‘clean’ surface for a piece of evidence. In this way, the lab benches are not in direct contact with the GSR containing pieces of evidence and potential transfer of particles from pieces of evidence is minimized. In addition to earlier work that reported on the presence of indicative particles on brown recycled paper, it was found that ‘clean’ unused paper can contain a large amount of particles containing the elements Pb, Ba, Ca, Si, Al, and P with various peak intensities. According to standard operating procedures in line with the ASTM standard, such an elemental compositions should be classified as indicative for gunshot residue. The presence of these kinds of particles can therefore have large implications in GSR investigations. Due to the large number of particles present in the paper, the particles are being transferred in large amounts contaminating the evidence itself and thus resulting in potential false positives.

During a shooting in a confined space, at least three shots were fired with a revolver, and the victim was shot through a door. As the suspect denied any contact with a firearm, the presence of GSR on the pieces of evidence (his trousers and a sampling of his car) were of prime importance in this case. The case was brought to trial by the public prosecutor after GSR particles were detected on the samples from the car. During the trial, the defense attorney successfully questioned the source of the GSR particles found in the car. The attorney argued that the GSR particles could also originate from a coffee shop as a colleague of the defendant used the car to visit one before the car was sampled. As a result, the court concluded that this activity could offer an alternative explanation for the source of the GSR particles and that they should be omitted as evidence. Following the appeal of the prosecutor, additional investigations were performed to study the prevalence and persistence of GSR particles in cars and coffee shops in order to determine the likelihood of this alternative source.

Benchtop Micromanipulator for Precise Sampling in the Forensic Sciences
Steven M. Barnett — Barnett Technical Services
In criminalistics, as well as in a range of other fields, handheld sample manipulation is challenging because over time, particles, fibers, and films of interest continue to decrease in size. A benchtop micromanipulator provides a simple way to perform many of these sample manipulations. A range of examples will be shown, including:

• isolation of short fibers from an adhesive surface
• isolation of glass particles from clothing
• separation of individual film layers from a multilayer paint structure
• scraping of oxide film from a knife
• isolation of sperm sample from clothing

Caught in the Act! Multispectral LANDSAT Imagery and SEM Identification of Potentially Toxic Fly Ash at Steam Power Plant Waste Disposal Sites
Wayne C. Isphording — University of South Alabama and Tulane University
The Alabama Department of Environmental Management (ADEM) and the U.S. Environmental Protection Agency (EPA) strictly regulate disposal of industrially generated toxic waste and requires its removal from sites where generation takes place. Permanent safe storage of such materials, similarly, is mandated and must be at locations deemed safe for projected long-term storage. One major source of such wastes are coal-fired, steam power plants used for generation of electricity. Historically, these have long been associated with power stations using coal, which produces a deleterious waste by-product in the form of fly ash. The cost of removing the large quantities of ash generated at power plants is sizeable, and to avoid this, ash has often been placed in on-site storage areas. Leaching of these waste sites by rainfall events has, in many cases, impacted adjacent water bodies that are used as sources of drinking water by nearby cities or towns and food sources derived from the resident aquatic fauna that are consumed by the local populace. As a consequence, most states now strictly prohibit on-site storage of fly ash.

Because of the large size of fly ash disposal sites, concealment is impossible, and they can be easily identified by their distinctive physical, mineralogical, and chemical properties. Even at an elevation of 700 miles above the Earth’s surface, their distinctive spectral signal is apparent on photos taken by the orbiting LANDSAT system. For example, samples collected from the Barry Steam Plant located 30 miles north of Mobile, AL possess abundant alumino-silicate and iron oxide spherules that are universally identified with fusion reactions associated with the high temperature burning of coal. These are easily visible in SEM photos of the fly ash and their distinctive chemical composition is further confirmed by EDS analysis. The ash at the Barry Plant also possesses a marked heavy metal signature that has produced a “mercury anomaly” not only in the discharge canal associated with the power plant, but also in the adjacent wetland area. The EPA has posted signs warning that consumption of fish from this area is harmful to health and should be avoided. Problems associated with a number of environmental restrictions (ash storage, chemical and thermal discharge exceedances, etc.) at six of the power stations resulted in the assessment of $1.25 million in fines against Alabama Power by the State in 2018. The company has acknowledged that planned conversion of their power plants to gas-fired systems will largely eliminate these problems.

Three Char and Soot Fire Cases
Andrew A. “Tony” Havics — pH2, LLC
Over the past two decades, restoration of fire- and smoke-impacted buildings has increased dramatically. In response to these impacts, inspection protocols and microscopical analysis methods have been devised by the Restoration Industry Association, in cooperation with the Indoor Environmental Standards Organization. The analysis can be supplemented by the ASTM method on soot, if so desired. Other techniques can also be used to help identify or supplement identification of sources of fire impact and the level of impact. A set of three fire cases involving the analysis of char and soot are used to illustrate the methods and techniques available in these cases. Two of these were from two-story residential properties and the third is from a multi-story commercial property.

Investigation into the Mechanism of Corrosion of a Pharmaceutical Glass Container
Richard S. Brown and Jake Spry — MVA Scientific Consultants
A glass container with surface corrosion on its internal diameter (ID) was examined using a combination of differential interference contrast microscopy (DIC), scanning electron microscopy coupled with energy dispersive X-ray spectroscopy (SEM-EDS), and analytical transmission electron microscopy coupled with energy dispersive X-ray spectroscopy (ATEM). After mapping potential corrosion sites, the containers were cut with a diamond saw to expose the glass container’s ID surface for additional direct examination by DIC and SEM-EDS. Selected corrosion pits were thin sectioned using a dual beam focused ion beam secondary electron microscope (FIB). The thin section was subsequently analyzed by AEM to analyze the corrosion surface in cross-section. Sub-surface modifications of the glass were apparent by imaging the thin-section by ATEM and extended well below the surface corrosion observed by DIC and SEM-EDS.

The Mystery of Leeuwenhoek’s Canoe
Brian J. Ford — Cardiff University
Recent publications in London have shone an unexpected light on the work of the pioneering microscopist Antony van Leeuwenhoek. It seems that he produced wooden paddles; while the unendingly fascinating investigation of his original microscopical specimens, discovered after more than three centuries by the speaker, has now been recast in a very different light. Illustrated with the latest video reports, this presentation will attempt to rectify some current misapprehensions.

FTIR Analytical Method for the Identification of Cellulose Fibers
Jonas Hoeg Thygesen and Anders Juul Lawaetz — Novo Nordisk Pharmatech, Koge, Denmark
Regulatory agencies call for the identification and characterization of any intrinsic, inherent, or extrinsic particles present in pharmaceuticals. Among the many tools for particulate and foreign material identification, Fourier-transform infrared (FTIR) microscopy has developed into one of the industry-standard workhorses. The common approach during FTIR microscopy includes measurement of the unknown fiber and comparison of the spectrum with a set of known reference spectra. This comparison is commonly based on correlation between the unknown and reference spectra. However, in the case of cellulose fibers, this approach does not allow distinction between, e.g., paper and cotton. Hence, the identification may stop once a particle has been identified as cellulose, thereby limiting the root cause analysis. This issue has been addressed at Novo Nordisk Pharmatech. Employing multivariate statistics, we have developed a method that allows us to discriminate between different cellulose fibers and to classify them into one of four groups: cotton, viscose/rayon, paper, and other cellulose fibers. This presentation will describe the knowledge gained during the development work and show how tools such as multivariate data analysis can be used to gain more insight from data already gathered.

Form Birefringence: Variable Birefringence?
Andrew A. “Tony” Havics — pH2, LLC
ASTM E2228 defines birefringence as “the numerical difference in refractive indices for a fiber, given by the equation: n|| – n⊥.” For crystals, it could be defined as the differences of refractive indices (RIs) of ε – ω for uniaxial crystals or γ – α for biaxial crystals. This is perhaps a simplification, as practical measurement of birefringence truly relates to the sum of four types of birefringence: intrinsic, form, strain, and circular. We will ignore circular birefringence for the time being and focus on linear birefringence. Most are aware of intrinsic birefringence and recognize it as being due to anisotropic periodicities in crystalline chain lattice that affect the velocity of linear polarized light. Many are also aware of strain birefringence, wherein stress modifies the polarizability of the molecules leading to a change of birefringence, typically from zero birefringence (isotropic) to some value of birefringence greater than zero. The concept of form birefringence has been limited in its description in texts and teaching materials for microscopy. Form birefringence could be described as the birefringence derived from a system of two periodically arranged components with anisometric forms with different RIs, where the size of the components must be small compared to the wavelength of light (< 500 nm). It comes in rodlet birefringence and layer or platelet birefringence. Form birefringence theory and practical examples help explain the phenomena and its observation in polarized light microscopy.

The Ever-Expanding World of Microscopy, Imaging, and Microanalysis
John A. Reffner — John Jay College, CUNY
Today, microscopy is being stimulated by the barrier of the diffraction limit of resolution being breached. The ability of scanning probes to reach nano-spatial resolution and the integration of microscopy with spectroscopy is a new technology. Confocal fluorescence microscopy and near-field optical scanning microscopy are capable of resolving molecular structure. Photo-thermal infrared spectroscopy has demonstrated sub-micrometer spatial resolution and high quality infrared absorbance spectra. Combining scanning probe with tunable laser technology or synchrotron radiation makes molecular vibrational spectra with nanometer scale spatial resolution possible.

The tentacles of microscopy extend to many disciplines and levels of complexity. The circus of life seen with a microscope in a drop of pond water has inspired many to pursue scientific careers. The hand lens gave us the means to explore the minutia of a fingerprint or discover the many minerals in rocks and sand. The pathologist’s diagnoses of diseases, by studying the microstructure tissue sections, play a vital role in public health. Metallurgists and material scientists use microscopy in many ways to improve materials or to determine why materials fail. Where will the new technologies take us? How will we become educated about these technologies and their application to real-world problems?

Plastics in a Refreshing New Light
Brian J. Ford — Cardiff University
The news is dominated by reports about the evils of plastic, and there is a rash of current books promoting the idea of a plastic-free future. Curiously, many plastics are biodegradable and can be metabolized by fungi, including Aspergillus and Penicillium, while expanded polystyrene has recently been shown to be a suitable foodstuff for insect larvae. Microbial polymers can offer us plastics for the future that are easily biodegradable, and even the massive drifts of plastic waste (like the Great Pacific Garbage Patch) could be usefully harnessed.

Unwanted Connections — from Whiskers to Nanotubes
James R. Millette — Millette Technical Consulting
Electrically conductive particles, especially those with elongated morphologies, are an important concern in areas housing electronic equipment such as data centers. A number of catastrophic computer system failures have been attributed to zinc whiskers reported from zinc-plated floor materials. Metal turnings and wear debris from carbon fiber products can also provide the opportunity for unwanted connections (short circuits) between electrical components. Most recently, ultra-microscopic ‘fiber’ nanotubes have caused concern about unwanted electrical connections on the smallest scale. Microscopy is the best tool to find these very small electrical connectors among the dust particles that inhabit electronic information storage systems. Elemental analysis from scanning electron microscopy coupled with energy-dispersive X-ray spectroscopy (SEM-EDS) is very useful for zinc and other elongated metal particles. Transmission electron microscopy coupled with EDS (TEM-EDS) is necessary for the investigation of nanotubes.

Andreas Sigismund Marggraf and the First-Time-In-History Use of a Microscope to Prove the Identity of a Chemical Substance
Jan Burmeister
The life of Andreas Sigismund Marggraf, an 18th century Berlin chemist, will be portrayed in light of his most notable and economically important discovery (published in 1749), that Beta vulgaris contains sugar, which is identical to ‘ordinary’ sugar from sugar cane of tropical origin. The use of the microscope to prove the identity of the two compounds will be outlined as the first time in history that such an attempt was successfully made.

Polymerography: Chemical Etching of Polymers
Andrew A. “Tony” Havics — pH2, LLC
There are many polymers for which etching techniques have been published. These include:
• polyolefins: polyethylene (LDPE, HDPE) and polypropylene (PPE)
• polycarbonates (PC)
• polylactide (PLA)
• natural rubber (NR)
• butadiene rubber (BR)
• nitrile rubber (NR)
• polyvinyls: polymethylmethacrylate (PMMA), polystyrene (PS), polyvinyl chloride (PVC), and polyacrylonitrile (PAN)
• styrene/acrylonitrile (SAN)
• acrylonitrile butadiene styrene (ABS)
• fluorocarbon polymers: polytetrafluoroethylene (PTFE), polyvinyl fluoride (PVF), and polyvinylidene fluoride (PVDF)
• polyethers
• polyesters: polyethylene terephthalate (PET)
• poly(aryl ether sulfone)s (PAESs)
• aliphatic polyamides (nylon)
• aromatic polyamides: aramids (Kevlar, Nomex)
• cellulose polymers (CELL)

This presentation will cover the theory behind polymer etching, followed by examples of what etching can reveal with light microscopy.

Continuing Adventures in Fluorescence
Charles Mazel — NIGHTSEA
NIGHTSEA primarily develops equipment for viewing and documenting fluorescence, both off-the-shelf and custom, and in doing so we encounter diverse opportunities and challenges. Some of these do not even involve fluorescence. This talk will review some of our experiences in the past year: what we have learned, what we are working on, and even how Inter/Micro itself proved invaluable in one project.

Force Feeding Physics
Brian J. Ford — Cardiff University
The current fashion for physics has encouraged the view that functioning of living microorganisms can be reduced to elementary constructs. However, many of the explanations under this Cartesian tradition are unduly simplistic, and their proponents fail to grasp the intricacies of living systems. In this presentation, we will contrast some current examples with the realities observed by microbiologists, proposing that greater effort should be taken to promulgate a more realistic interpretation of life under the microscope.