Spectral emission variations as a function of time post serum starvation.

(A) 3D scatter plot was generated by utilising cursors (size = 0.005) to select wavelengths and widths in the phasor plot corresponding to each time course (control: n = 274, 10 min: n = 471, 20 min: n = 519, 30 min: n = 439, 40 min: n = 370). The number of data points collected (n) depended on the shape of the phasor; a condensed phasor resulted in lower ‘n’ value, conversely a more spread out phasor resulted in a higher n value. (B-i) The phasor shape of emissions corresponding to the cell after the removal of background emissions. (B-ii) The phasor shape of emissions corresponding to the background after the removal of the cell emissions. Relative to the cellular emissions, background emissions were apparent near the centre of the plot and exhibited broader widths. (C) A different coloured cursor was utilised to select the most intense region of the phasor plot for each time course, and the emissions were mapped to the original fluorescence images to generate spectral images.