0.1 × 106 cells were seeded onto 25 mm diameter MgF2 windows (Global Optics) in a 6-well plate and incubated for 24 hours. Cells were treated with the relevant photosensitiser in serum-free DMEM at the appropriate concentration and duration (1 hour, 100 ng/mL for Verteporfin; 3 hours, 10 mM for 5-ALA; 24 hours, 10 ng/mL for Temoporfin) and were then fixed with 4% paraformaldehyde (PFA) solution for 20 minutes at room temperature and stored at 4 °C in PBS until required for imaging. Cell spectra were obtained from cells in PBS using a 63× water-immersion objective lens and a 100 μm fibre acting as a confocal pinhole on a confocal Raman microscopy setup (Witec GmbH). Raman spectra were obtained using a 785 nm laser (Toptica Extra) with a power of ~80 mW and a 10 second integration time. For each cell, 5 spectra were obtained from random locations within the cell. Spectral processing was performed in MATLAB (2017b) using scripts developed in-house. First, the spectra were cropped to between 510 cm-1 and 1825 cm-1 and the fluorescence background was removed using a Whittaker filter baseline subtraction (λ = 100,000). Cosmic ray peaks present in the spectra were removed and the spectra were smoothed using a 1st order Savitzky-Golay filter with a frame width of 7. Partial least squares-discriminant analysis (PLS-DA) was performed using PLS Toolbox (Eigenvector Research) within the MATLAB environment. Pre-processed, normalised (area under the curve), and mean-centred spectra were classified using PLS-DA performed with 6 latent variables and a Venetian blinds cross-validation with 10 data splits.

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