Fig 1: Optical redox ratio (ORR) of cells and solutions with different acquisition parameters. Human pancreatic cancer cells (MIA PaCa-2, ATCC CRL-1420) in DMEM with 10% FBS and 1% Penn/Strep antibiotic were imaged using a custom epi-detection multiphoton imaging system with 80 MHz excitation from a tunable fs laser (Insight X3+, Spectra Physics). Sequential images of FAD and NAD(P)H autofluorescence in cells were acquired using different excitation wavelengths and objective lenses, as indicated above each image. Images of 1 mM NADH and FAD solutions were additionally acquired at all acquisition and illumination conditions. ORR was calculated from FAD and NAD(P)H intensities (fifth row) or calculated with normalized intensities (sixth row) by dividing by the mean intensity of 1 mM NADH and FAD at the corresponding acquisition and illumination conditions. Images are sums of 20 frame averages at the same location, with 20 mW incident power on the sample (incident light pulse width = 170 fs; pixel dwell time=2.5 μs; frame rate = 0.19 fps, frame size=512×512 pixels/100×100 μm). Images were collected using hybrid detectors at constant gain for all measurements. Cell and nuclear areas were masked using an arbitrary threshold NAD(P)H intensity at 740 nm and that mask was used for all images from the same objective lens. Raw images from both channels were summed with a 3×3 spatial filter prior to ORR calculation, and ORR images were median filtered with a 5×5 filter to remove outliers. Note that NAD(P)H signal leaks into the FAD channel when FAD is acquired at 740 and 750 nm, which leads to a washing out of features in the ORR images. No spectral unmixing was used to account for the NAD(P)H leakage into the FAD channel but should be considered for future work.