Understanding and classifying inherent tumor heterogeneity is a multimodal approach, which can be undertaken at the genetic, biochemical, or morphological level, among others. Optical spectral methods such as Raman Spectroscopy aim at rapid and non-destructive tissue analysis, where each spectrum generated reflects the individual molecular composition of an examined spot within a (heterogenous) tissue sample.
Using a combination of supervised and unsupervised machine learning methods as well as a solid database of Raman spectra of native glioblastoma samples, we succeed not only in distinguishing explicit tumor areas - vital tumor tissue and necrotic tumor tissue can correctly be predicted with an accuracy of 76% - but also in determining and classifying different spectral entities within the histomorphologically distinct class of vital tumor tissue. Measurements of non-pathological, autoptic brain tissue hereby serve as a healthy control since their respective spectroscopic properties form an individual and reproducible cluster within the spectral heterogeneity of a vital tumor sample. The demonstrated decipherment of a spectral glioblastoma heterogeneity will serve valuable especially in the field of spectroscopically guided surgery to delineate tumor margins and to assist resection control.