Quantitative FLIP and single particle tracking of mobile IB’s in the cytoplasm. A FLIP experiment with a bleach region of 10 pixels in diameter was performed in CHO cells expressing eGFP-Q73 on a temperature-controlled stage of a confocal microscope maintained at 35 ± 1°C. The total frame rate was 1.45 sec. A, montage of every 20th frame of the data. Arrows point to an inclusion body (IB) in the cytoplasm. B-E, pixel-wise fit of the data to a StrExp function with B, amplitude map; C, map of the stretching parameter; D, time constant map; E, χ2-values showing the quality of the regression. All values are color-coded using a FIRE-LUT, and the range is indicated by the respective color bar. The time constant is given in seconds; all other values are without units. F, first frame of the time series is shown in green, while selected subsequent frames are overlayed in red (box in F shows movement of the IB). G, FL of eGFP-Q73 in the IB (red symbols) with fit to the StrExp function (black line). H, x,y-plot of the trajectory of the IB during the FLIP experiment. I, mean square displacement (MSD) calculated from the trajectory and linear fit to the five intital data points (red line). J, K, simulation and tracking of a particle experiencing FL with a rate constant, k = 0.01 sec-1. J, trajectory separated in x- (straight lines) and y-direction (dotted lines) for a particle undergoing bleaching (black lines, simulated trajectory; red lines, tracked trajectory). The tracked trajectory coincided with the simulated trajectory until to 180 sec (or frames). K, intensity of the tracked particle (grey lines) compared to a mono-exponential fit with residual. Bar, 5 μm.