Figure 4

Barrier-limited FLIP of eGFP shuttling between nucleus and cytoplasm. McArdle RH7777 cells expressing eGFP in the cytoplasm and nucleus were placed on a temperature-controlled stage of a confocal microscope maintained at 35 ± 1°C. A 30 pixel diameter circular region in the cytoplasm (white circle) was repeatedly bleached with full laser power, while the whole field was scanned with 0.5% laser output between the bleach scans such that the total frame rate was 2.6 sec. A montage of every 30^th frame of the data (upper panel, ‘data’) or of the reconstruction from a pixel-wise fit of data to the StrExp function (lower panel, ‘fit’). B, amplitude map; C, background map, each with three boxes numbered 1 to 3. D, FL in these three boxes along the stack (colored symbols) + fit to StrExp function (colored lines). E-H, parameter maps produced by PixBleach for: E, the stretching parameter (see Eq. 1; the color code for h = 1, the mono-exponential case, is given in small box); F, χ²-values showing the quality of the regression; G, time constant map; H, number of iterations. All values are color-coded using a FIRE-LUT, and the range is given below the images with a color bar. The time constant is given in seconds; all other values are without units. I, J; histograms of the stretching parameters (I) and the time constants (J) for the cell, shown in A-H. Bar, 5 μm.