Effects of tissue fixation on Raman spectroscopic characterization of retina

Raman spectroscopy is a non-invasive and non-destructive tool that has been widely applied in Agricultural and Bio-system Engineering field. It provides a unique “fingerprint” to different chemical, and structural, information at molecular level. Acquisition of Raman spectrum requires a relatively short collecting time and minimum sample preparation, which makes Raman spectroscopy an attractive method for characterizing biological samples. Since retina is the most exposed part of the central nervous system, irregular protein transformation, or accumulation caused by diseases, can be found at an early time in this area. Because of this reason, Raman spectroscopy is often utilized to analyze samples from specific layers in retina to detect certain diseases, such as glaucoma, Parkinson’s disease, and Prion related diseases at early stages. In order to have a better preservation of tissue architecture and longer storage time, collected retina samples are commonly first fixed by chemical fixative. Typical fixatives include: Modified Davidson’s fixative, Bouin’s fixative, Ethanol fixative or conventional Formalin fixative. After fixation, samples are then embedded with paraffin to allow for fine sectioning. Every fixating protocol has different ingredients, and there is no universal fixative that is ideal for all the tissue types. Since fixation introduces chemical modification to the tissue samples, it is important to understand what changes it may cause to the Raman spectroscopic characterization of the tissue samples. For best result, four fixation methods are studied and analyzed in this research. The purpose is to identify a mathematical methodology which can be used to remove the background noise produced by each chemical fixative, and to determine how each fixative alters the collected Raman spectra, and in which regions alternation appears. Although this sounds theoretically promising, the actual cellular