Fig. 2: Penalized Reference Matching Method.
a A lipid reference spectrum (blue) was collected by spontaneous Raman spectroscopy, and an SRS HSI pixel spectrum (orange) was compared with the reference spectrum. b Demonstration of shifts in pixel’s spectrum: 7.13 cm−1 left shift and 21.39 cm−1 right shift. These positional offsets decrease the final similarity score because the penalty term scales exponentially to the positional offset. c Matching scores are retrieved by dot product between the reference signal and SRS pixel’s signal. Then the penalty, estimated with a quadratic function, is subtracted from each matching score. When the shift wavenumber is high, a high penalty will be given. The highest value in this score curve will be used as the similarity score between this pixel spectrum and that of the pure reference standard. d Image illustrating the distribution of cardiolipin in a murine dentate gyrus sample with a penalty coefficient of α = 1 × 10−4. e Image with the penalty coefficient α = 4 × 10−4. The full range of the cardiolipin distribution was not clearly shown due to over-penalization. f An over-saturated image with the penalty coefficient α = 0.25 × 10−4. Almost all pixels have a high similarity score due to under-penalization. Scale bar, 100 µm. HSI Hyperspectral Image.
