TY - THES ID - 134545879 TI - Development of optical clearing protocols for the detection of micro- and nanoplastics in biological samples AU - Vandermeulen, Teun AU - Roeffaers, Maarten AU - KU Leuven. Faculteit Bio-ingenieurswetenschappen. Opleiding Master in de bio-ingenieurswetenschappen. Biosysteemtechniek (Leuven) PY - 2023 PB - Leuven KU Leuven. Faculteit Bio-ingenieurswetenschappen DB - UniCat UR - https://www.unicat.be/uniCat?func=search&query=sysid:134545879 AB - As society becomes more reliant on the use of plastics, consumption grows every year and waste increases proportionally. Plastics are released in nature, polluting the environment, biological life, and ecosystems. Once discarded, plastics degrade to microplastics (<5 mm) and nanoplastics (<1 µm) due to weathering processes and photodegradation. Micro- and nanoplastics (MNP’s) can enter the human body via several pathways, and induce toxicological effects on human health. Very little is known regarding these adverse effects due to a lack of appropriate detection techniques. Also, localization of particles in biological samples is required to investigate local effects, meaning that the preservation of bio-context is essential to achieve this. Staining of MNP’s with Nile Red (NR), a solvatochromic dye, can serve as a fluorescent labeling technique for MNP’s, enabling high-resolution particle detection and polymer identification. However, since NR has a high affinity for lipophilic carbon structures, organic materials like lipids get co-stained, inducing false-positive results. Furthermore, biological samples often are opaque, limiting laser transmission due to light scattering. Thirdly, autofluorescence causes a lot of background signal, interfering with the signal obtained from small plastics and making detection of MNP’s more challenging. For this reason, optical clearing protocols are needed to resolve these issues by extracting these structures from biological samples. 3D spheroids (co-cultured with MNP’s) and tissue samples were prepared and anchored in an acrylamide gel. This enabled the preservation of bio-context, allowing particle localization in a subsequent stage. Next, optical clearing steps were added and different protocols were compared, starting from a protein digestion step. This resulted in a more effective removal of lipids, which enhanced laser transmission (decrease in scattering) and reduced autofluorescence. This facilitates future research towards MNP-detection with NR staining, as false positive results get ruled out more effectively. ER -