Fig. 4. (top) Time-lapse imaging of PYP WT–BFP–NLS and PYP 4R–BFP–NLS expressed in HEK 293T cells with AcFCANB. The images were collected every 10 min after the addition of the probe (2 μM), with the excitation at 473 nm by using a 490–590 nm emission filter for AcFCANB, and with the excitation at 405 nm by using a 420–520 nm emission filter for BFP. (bottom) Labeled fraction of PYP against incubation time (N = 3). The quantification was conducted by selecting cells exhibiting the equivalent level of fluorescence of BFP at T = 0 min. Scale bar: 10 μm.