ARTD10 shuttles between the cytoplasmic and nuclear compartments. A. Schematic of the iFLAP imaging approach. Full length ARTD10 or different fragments of ARTD10 were fused N-terminally to CFP-YFP. The proteins were transiently expressed in COS7 cells. The YFP and CFP channels of the confocal microscope were adjusted so that the YFP and CFP fluorescence of the CFP-YFP-ARTD10 constructs appeared with almost identical signal intensities (iYFP ≅ iCFP). For each pixel of the image, using the formula intensity = 1–(iYFP/iCFP), a new image was calculated with almost zero signal intensity depicted in blue. Selective bleaching of YFP using the 514 nm laser of the confocal microscope within the rectangular bleaching-ROI (ROI = region of interest) generates a subpopulation of CFP-YFP-ARTD10 molecules that are only detected in the CFP-channel. These molecules generate a signal in the processed data channel. According to their intensities these signals are depicted in rainbow colors with blue representing weak and red strong signals. They diffuse or are transported through the cytoplasm and, depending on the protein analyzed, appear within the nucleus. A non-bleached cell is always included to demonstrate that signals are not generated as a result of bleaching upon image acquisition. B. The indicated proteins were expressed transiently in COS7 cells. A steady-state distribution of the different CFP-YFP-fusion proteins is shown in the panel on the left. The subcellular distribution of bleached fusion proteins is shown at different time points after bleaching. For control identical cells are shown prior to bleaching (pre-bleach). The scale bar indicates 10 μm.