Author ORCID Identifier

Defense Date


Document Type


Degree Name

Master of Science


Forensic Science

First Advisor

Sarah J Williams

Second Advisor

Tracey Dawson Cruz

Third Advisor

Joseph E Reiner


Current methods of mixture separation in forensic DNA laboratories typically deconvolute the mixture after analysis using statistical analysis or probabilistic genotyping. To save time and effort of labs already backlogged, a method to separate mixtures on a cellular level before analysis needs to be developed. Optical trapping is a method that uses a focused 1064 nm laser to manipulate cells. Previous research has shown that approximately 50 spermatozoa or 15 leukocytes from a liquid sample are required to produce a full STR DNA profile. It was found that the number of spermatozoa required remains constant when the method of sample collection is changed to cotton swab mimicking sexual assault evidence. These spermatozoa can be collected at a rate of approximately two cells per minute and can retrieve nearly all spermatozoa within 400 nL if the surface of the coverslip upon which trapping occurs is modified with poloxamer 407. While leukocytes isolated from whole blood can be trapped, it has been found that leukocytes that have been reconstituted from a swab lose their morphology and are hard to identify and trap. To combat this issue, DAPI was used to stain the nucleus of leukocytes to identify them under microscopy for trapping. Stained cells were easily identified but were repelled by the trap. This research demonstrates that optical trapping is an efficient method to separate spermatozoa even in samples collected from sexual assault kits with low numbers of spermatozoa to generate full, single-source STR profiles. Further research should be conducted on the best method to separate reconstituted leukocytes of irregular morphology.


© Benjamin J O'Brien 2020

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