for both unlabeled and SNBD-labeled peptides were calculated using the software Molinspiration. B) Representative CD spectra of unlabeled TAT, penetratin, and sC18 (black) and their SNBD-labeled counterparts (green). Spectra were recorded with 50 µM peptide in 30% trifluoroethanol in aqueous buffer (100 mM NaF, 10 mM KH2PO4 pH 7.5), and obtained at 0.5 nm intervals between wavelengths of 180 nm to 260 nm and averaged from 3 scans. Background signals were subtracted and the signals were recorded as millidegrees at 25 °C. Mean residue ellipticity (MRE) was calculated according to [θ]MRE = θ/(c × l × Nr), where θ is the recorded ellipticity in millidegrees, c is the peptide concentration in dmol•L -1 , l is the cell path length in cm, and Nr is the number of residues. Cartoon representations of the secondary structures indicated by the CD spectra are depicted in grey, alongside the percentage of helicity (when applicable) for the unlabeled CPPs (grey) and labeled CPPs (green). The helical percentage (Hα) was calculated from the Luo-Baldwin formula. Wash-free imaging of CPP trafficking in live cells. Having synthesized native-like fluorogenic analogues of three different CPPs, we conducted wash-free microscopy experiments in live HeLa cells as a well-established line for imaging studies of cellular internalization. The three labeled CPPs were added to the cell medium, and their fluorescence localization was monitored over the course of 20 minutes without any washing steps and using LysoTracker Red as a subcellular marker. TAT-SNBD showed internalization by direct translocation (Figure ) with high accumulation across the cell membrane, and, to a lesser degree, diffusion throughout the cytoplasm over time (Figure , Figure and Movie S1).