1 × 106 A549 cells were seeded into each of the wells of a 6-well plate in serum-supplemented RPMI and incubated for 24 hours at 37 °C and 5% CO2. Cells were then incubated with serum-free DMEM with or without 10 mM 5-ALA for 3 hours at 37 °C and 5% CO2. Solutions were then removed, and cells washed with DPBS. Serum-free DMEM was then added to all cells. Cells in each well were illuminated using the multimodal optical probe and associated lasers for Raman spectroscopy and PDT. The probe was placed underneath the centre of each well and held in place by a support stand. Laser outputs were adjusted to 100 mW and cells illuminated for 120 seconds with one of the lasers (no laser illumination for control cells) before the plate was returned to the incubator for 24 hours at 37 °C and 5% CO2. After 24 hours incubation, a LIVE/DEADTM assay was performed using a 2 μM calcein AM (Thermo Fisher Scientific) and a 4 μM ethidium homodimer-1 (Thermo Fisher Scientific) solution in DPBS. Media was removed from the cells and cells were washed with DPBS. LIVE/DEADTM reagent was added to the cells and incubated for 30 minutes at room temperature. Reagent was removed from cells and cells washed with DPBS. Cells were then placed in DPBS for immediate imaging. Imaging was performed using an Olympus IX71 inverted fluorescence microscope with a 4× objective. Multiple images were manually stitched together for Figure Figure33A.

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