Fig. 5.

Single-pixel kinetic analysis of Rhodamine B uptake by MCF7 cells. (a) ntensity and (b) fluorescence lifetime of four selected pixels imaged at 25 frames per second (fps) during 1 min of acquisition. Pixel locations within the image are marked in (c), an image of the average multiphoton intensity of the final 500 frames in the acquisition. The pixels chosen represent four locations with different intensity and lifetime profiles due to spatial and biochemical differences: extracellular space outside of the cell cluster (red), cell cytosol close to the extracellular space (green), nucleus of the same cell (blue), and cell cytosol closer to the interior of the cell cluster (purple). (d) Calculated rate constant for intensity kinetics for each pixel. (e) Calculated rate constant for fluorescence lifetime kinetics for each pixel. Scale bar represents 20 µm.