Unraveling Molecular Composition in Biological Samples-Systematic Evaluation of Statistical Methods for the Analysis of Hyperspectral Raman Data

Recently, confocal Raman microscopy has gained popularity in biomedical research for studying tissues in healthy and diseased state due to its ability to acquire chemically selective data in a noninvasive approach. However, biological samples, such as brain tissue, are inherently difficult to analyze due to the superposition of molecules in the Raman spectra and low variation of spectral features within the sample. The analysis is further impeded by pathological hallmarks, for example beta-amyloid (A beta) plaques in Alzheimer's disease, which are often solely characterized by subtle shifts in the respective Raman peaks. To unravel the underlying molecular information, convoluted statistical procedures are inevitable. Unfortunately, such statistical methods are often inadequately described, and most natural scientists lack knowledge of their appropriate use, causing unreproducible results and stagnation in the application of hyperspectral Raman imaging. Therefore, we have set out to provide a comprehensive guide to address these challenges with the example of a complex hyperspectral data set of brain tissue samples with A beta plaques. Our study encompasses established as well as novel statistical methods, including univariate analysis, principal component analysis, cluster analysis, spectral unmixing, and 2D correlation spectroscopy, and critically compares the outcomes of each analysis. Moreover, we transparently demonstrate the effect of preprocessing decisions like denoising and scaling techniques, providing valuable insights into implications of spectral quality for data evaluation. Thereby, this study provides a comprehensive evaluation of analysis approaches for complex hyperspectral Raman data, laying out a blueprint for elucidating meaningful information from biological samples in chemical imaging.