Imaging was done using an Olympus IX-81 inverted microscope as has been described previously [24] (link). Fluorescence images were acquired at 10 ms integration time using a 100 W Hg arc lamp, a 470/40 nm bandpass excitation filter at 150X magnification with a 1.45 NA objective (Olympus) focusing on apical membranes of the lamella of the cells. Detection of all colors was done simultaneously through a QuadView emission splitter (dichroic mirrors at 585, 630, and 690 nm, and emission bandpass filters 535/30, 605/20, 655/20, and empty position) and an Andor EMCCD camera at 25 Hz. The camera has a pixel size of 16 µm, such that the projected pixel size in our case was 107 nm. The spectral overlap of the QD605, QD655, and QD705s among the image channels is such that less than 5% of the QDs are detected in the wrong image channel [24] (link). Image acquisition was controlled by Andor IQ software and movies of 1200 frames (∼48 s) were recorded at RT. The signal-to-noise in the image channel of the YFP-KRas2 fusion protein under the chosen imaging conditions (10 ms camera integration, 25 Hz imaging rate) is very low on an image frame by frame basis. Hence we have so far used these images only to provide a detailed image of the footprint of the plasma membrane of each cell for the duration of the time lapse sequence by generation of a Sum Intensity Projection image in ImageJ.
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