Editing Forensic palynology (section)

== Methods ==

=== 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.<ref name=":2">{{Citation |last1=Bock |first1=Jane H. |title=Chapter 10 – Additional Approaches in Forensic Plant Science |date=2016-01-01 |url=https://www.sciencedirect.com/science/article/pii/B9780128014752000105 |work=Forensic Plant Science |pages=129–147 |editor-last=Bock |editor-first=Jane H. |place=San Diego |publisher=Academic Press |language=en |isbn=978-0-12-801475-2 |access-date=2022-03-02 |last2=Norris |first2=David O. |editor2-last=Norris |editor2-first=David O.}}</ref> Samples then need to be prepared and placed on slides in order to fully be safe from contamination.<ref name=":2" /> 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.<ref name=":3">{{Cite journal |last1=Mildenhall |first1=D. C. |last2=Wiltshire |first2=P. E. J. |last3=Bryant |first3=V. M. |date=2006-11-22 |title=Forensic palynology: Why do it and how it works |url=https://www.sciencedirect.com/science/article/pii/S0379073806005007 |journal=Forensic Science International |language=en |volume=163 |issue=3 |pages=163–172 |doi=10.1016/j.forsciint.2006.07.012 |pmid=16920303 |issn=0379-0738}}</ref> 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.<ref name=":3" /> Due to the lack of palynologists in the forensic field, other forensic scientists that are present may have to collect the samples.<ref name=":3" /> This raises issues in terms of the quality of the sample, since collection sites for the sample should be determined depending on the case.<ref name=":3" /> Discussion with the investigation team is necessary in order to establish the best sampling method.<ref name=":3" />

When collection sites have been determined, samples can be retrieved with clean instruments and placed into tightly sealed, sterile containers.<ref name=":3" /> Examples are "sterile zip-lock plastic bags, or screw-top plastic (in preference to glass) containers."<ref name=":3" /> After each sample, instruments should be thoroughly cleaned or replaced to prevent contamination.<ref name=":3" /> In cases, where collection is by hand, gloves should be used and replaced after each sample.<ref name=":3" /> Samples should be labeled and sample history documentation should be maintained to keep track of the people who have had access to the sample.<ref name=":3" />

=== 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.<ref name=":2" />

Samples are chemically processed with a mix of acids, sodium hydroxide, acetic anhydride with water washes in between.<ref name=":5">{{Cite journal |last1=Hawksworth |first1=David L. |last2=Wiltshire |first2=Patricia E. J. |last3=Webb |first3=Judith A. |date=2016-07-01 |title=Rarely reported fungal spores and structures: An overlooked source of probative trace evidence in criminal investigations |url=https://www.sciencedirect.com/science/article/pii/S037907381630069X |journal=Forensic Science International |series=Special Issue on the 7th European Academy of Forensic Science Conference |language=en |volume=264 |pages=41–46 |doi=10.1016/j.forsciint.2016.02.047 |pmid=27017083 |issn=0379-0738}}</ref> They are then neutralized, and the extracts are stained and mounted onto slides for microscopic examination.<ref name=":5" /> This helps in identification with the help of available reference collections to make comparisons on the pollen's characteristics.<ref name=":2" /> 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.<ref name=":4" />

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.<ref name=":4" />

DNA Barcoding is another method used to differentiate between pollen grains by comparing their DNA sequences.<ref name=":4" /> A pollen grain of 10 micrometers in length is required.<ref name=":4" /> 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).<ref name=":4" /> HTS is faster and less expensive than traditional methods for DNA barcoding.<ref name=":4" />