Figure 14.

Fluorescence anisotropy time-lapse imaging. (a,b) Fluorescence intensity (a) and weighted fluorescence anisotropy (b) time-lapse imaging (30 min) of HT1080 cells incubated in PARPi-FL (1 µM) for 1 min, during the course of the washing phase. Anisotropy images allow the researcher to follow target engagement in real time. An anisotropy threshold is assigned to distinguish among the different bound and unbound states. As a function of time, we observe a decrease of nonspecifically bound drug in the cytoplasmic region, whereas the bound fraction of the drug increases in the nucleoli, where PARP accumulates. The weighted fluorescence anisotropy images are color-mapped using the weighted RGB color-mapping scheme illustrated in the Experimental design section. Scale bars, 13 µm.