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Brillouin imaging relies on the reliable extraction of subtle spectral information from hyperspectral datasets. To date, the mainstream practice has been to use line fitting of spectral features to retrieve the average peak shift and linewidth parameters. Good results, however, depend heavily on sufficient signal‐to‐noise ratio and may not be applicable in complex samples that consist of spectral mixtures. In this work, we thus propose the use of various multivariate algorithms that can be used to perform supervised or unsupervised analysis of the hyperspectral data, with which we explore advanced image analysis applications, namely unmixing, classification and segmentation in a phantom and live cells. The resulting images are shown to provide more contrast and detail, and obtained on a timescale ∼10 2  faster than fitting. The estimated spectral parameters are consistent with those calculated from pure fitting.

Multivariate analysis of Brillouin imaging data by supervised and unsupervised learning