Spectral variation with respect to distance from cytoplasmic membrane.

Regions of the spectral image along the transect line (A) at 1 μm increments were isolated and 50 cursors (size = 0.005) were utilised to select emissions on the phasor plot corresponding to the selected regions of interest. (B) The variance of emissions appeared greater in the background (indicated by the largest circle) than the cytoplasmic membrane which was more variable than emissions found within the cell. (C) Removing the background emissions made apparent the difference in the degree of variability between the cytoplasmic membrane emissions compared to regions within the cell. (D) Within the population of emissions found 1–8 μm within the cell, each region’s emissions appeared to converge in non-overlapping parts of the spectrum; however, these λmax did share similar spectral widths. Note that the colour code for each scatter plot changes for each cell region from (B) to (D) as the background and cytoplasmic membrane emission are removed. White dotted line represents the nucleus and the red dotted line represents the nucleolus.