Sample Collection

Because pollen can be easily picked up by anyone, it is important that pollen samples are collected as soon as possible to prevent contamination from outside sources.^[19] Samples then need to be prepared and placed on slides in order to fully be safe from contamination.^[19] The process of preparing the samples and identifying them is time-consuming. When collecting a sample, it should be paired with site surveys and photos of the scene to provide context for later uses.^[1] For example, if the pollen evidence is used in court, then the additional context would be useful.

Sample collection methods will vary depending on the case investigation and on the collector.^[1] Due to the lack of palynologists in the forensic field, other forensic scientists that are present may have to collect the samples.^[1] This raises issues in terms of the quality of the sample, since collection sites for the sample should be determined depending on the case.^[1] Discussion with the investigation team is necessary in order to establish the best sampling method.^[1]

When collection sites have been determined, samples can be retrieved with clean instruments and placed into tightly sealed, sterile containers.^[1] Examples are "sterile zip-lock plastic bags, or screw-top plastic (in preference to glass) containers."^[1] After each sample, instruments should be thoroughly cleaned or replaced to prevent contamination.^[1] In cases, where collection is by hand, gloves should be used and replaced after each sample.^[1] Samples should be labeled and sample history documentation should be maintained to keep track of the people who have had access to the sample.^[1]

Analysis

Analyzing the samples, once the palynomorphs have been extracted, will allow for identification, which can then be used in a forensic case to relate a person or object to a crime scene, or even to determine whether the scene at which the pollen was found was the primary or the secondary scene.^[19]

Samples are chemically processed with a mix of acids, sodium hydroxide, acetic anhydride with water washes in between.^[17] They are then neutralized, and the extracts are stained and mounted onto slides for microscopic examination.^[17] This helps in identification with the help of available reference collections to make comparisons on the pollen's characteristics.^[19] The scanning electron microscope (SEM) has been used traditionally since the 1970s for primary identification of palynomorphs, but is very time-consuming, tedious, and not ideal for routine analysis.^[5]

Compared to the SEM, semi-automated pollen grain imaging techniques such as Transmitted Light Microscopy (TLM), Widefield fluorescent method, and Structured illumination (Apotome) were found to have a higher speed and accuracy when it came to the identification of pollen spores.^[5]

DNA Barcoding is another method used to differentiate between pollen grains by comparing their DNA sequences.^[5] A pollen grain of 10 micrometers in length is required.^[5] Once the sample is collected and prepared, genetic markers are placed, then the DNA is isolated, and finally the DNA is sequenced, usually through high throughout sequencing (HTS).^[5] HTS is faster and less expensive than traditional methods for DNA barcoding.^[5]

Austria, 1959

One of the earliest document cases in which pollen plays a key role took place in Austria. A man went missing, and was presumed murdered, but no body was found. The authorities had arrested a suspect, who had motive for the murder, but did not have a body or confession, and the case stalled. A search of the suspect's belongings yielded a pair of muddy boots. The mud was sampled and given to Wilhelm Klaus, at the University of Vienna's Paleobotany Department, for analysis. Dr. Klaus found modern pollen from a variety of species, including spruce, willow, and alder. He also found fossilized hickory pollen grains, from a species long extinct. There was only one area of the Danube River Valley that hosted those living plants, and had Miocene-aged rock deposits that would contain the fossilized species. When the suspect was presented with this information, he willingly confessed and lead authorities to the sites of both the murder and the body, both of which were inside the region indicated by Dr. Klaus.^[6][24]

United States, 1970

The first cases that involved forensic palynology in the United States was in 1970, where Honeybee pollination studies were issued. During this time honey pollen analysis began growing as the United States Department of Agriculture (USDA), insured beekeepers a higher world market price for their honey. Under the premisses that the honey was produced on USA grounds. Thus, honey samples were sent for pollen analysis, where confirmation that the honey was produced in the USA was concluded or not. Generally, most of the cases during this time involved lawsuits concerning beekeepers. For example, a Michigan beekeeper arose suspect of importing beehives from the southeastern USA that weren't inspected for mites. This led to the USDA inspecting honey samples where it was concluded that they were imported from the Southeastern region of USA. Due to that the honey contained floral types common to the Southeastern region and not found in Michigan.^[25]

United Kingdom, 1993

An example concerning Forensic Palynology in the United Kingdom, took place during 1993 handled by Patricia Wiltshire.^[26] Where it involved a murder case in which the body was laid on soil that preserved pollen. Wiltshire then found traces of walnut pollen in the soil and suspect's shoes, however, the walnut pollen found was unusual as there was no walnut site nearby. However, It was later discovered that a walnut tree was cut down thirty years before and the walnut pollen remained. the pollen was then analyzed and linked to the suspect in the crime scene. Thus, the walnut pollen provided a significant role solving the case.^[27]

New Zealand, 2005

After a home invasion, two burglars brushed past a Hypericum bush outside of the house. One of the burglars was brought in as a suspect, but all evidence was circumstantial, and the man did not confess. Analysis of his clothes revealed the Hypericum pollen. The presence of pollen is ubiquitous, but in this case, the pollen was clumped onto the clothing (rather than dusted) and did not seem to be simply the result of air dispersal. It was ultimately concluded that "the clothes had so much Hypericum pollen on them that they had to have been in direct and intimate contact with a flowering bush."^[8]

United States, 2015

A modern application of forsenic palynology occurred in 2015, in the city of Boston, Massachusetts. A body of a young female child was discovered by law enforemcent in the Boston Harbour, but no identifying features remained as the body was in the late stages of decomposition. Investigators submitted samples taken from the clothes of the victim, a blanket found with the body, as well as a small amount of recovered hair to the U.S. Customs and Border Protection directorate laboratory in Houston, Texas, for pollen analysis. The report from the submitted samples provided investigators with information they could use to identify the unknown victim.^[28]

The pollen assemblage created from the submitted samples indicated the vicitm was in the north-eastern United States before her death. The individual taxa of plant species observed in the assemblage also indicated that the victim lived in, or spent much of her time in, a developed, urban environment. The assemblage also captured pollen of the Lebanese cedar tree (Cedrus libani), native to the Eastern Mediterranean region of Europe. The species of cedar observed in the assemblage was thought by investigators to most likely be from an ornamental piece in a park or other conservation area. This is when the investigators discovered individuals of the Lebanese cedar tree in the Arnold Arboretum, a public park that is a part of Harvard University.^[28]

Investigators then asked around the neighborhoods surrounding the arboretum, and a tip led them to a resident who, after questioning, admitted that her boyfriend at the time had abused the child, which resulted in the child's death. The man who murdered the child was sentenced to serve a minimum of 20 years for second degree murder. The mother's involvemnt in the crime is not reported, though she served 2 years probation for accessory after a plea deal for providing information about her then boyfriend who had committed the act.^[28]

Argentina, 2023

A man was reported missing to the local police in Buenos Aries, Argentina. The family reported a possible suspect due to the fact the missing person and the suspect had intense arguments not long before his disappearance. The police searched the suspects house and found bloodstains on clothing items under the bed. They then took clothing items of the suspect for evidence analysis and soil samples from the garden of the suspects house. 25 days later, the police found the missing persons body was found 1000 meters off a coastline. Forensic mycology and palynology was then applied to analysis the palynomorphs and fungal species, as soil samples from the location of the corpse were analyzed. Samples were first acquired by scarping the surface area and then filtration and acetolysis was conducted to isolate the pollen samples. The analysis of the suspects clothing was then compared to the analysis of the corpse and the surrounding area. Results indicated that the corpse had a palynomorph composition of marine species, the quantity and exact species were all documented. The soil from the suspects house had a continental palynomorph composition that was also fully documented and recorded. The suspects bloodstained clothing had the same marine palynomorph composition as the body and the surrounding area. Soil samples in the suspects car and under the soles of the suspects shoes also contained the same palynomorph composition as the coastline area where the body was found. Along with other forensic evidence, the suspect was found guilty and the use of forensic palynology was used to help close this case.^[29]