To further increase the spatiotemporal resolution of cell internalization and trafficking, we explored the compatibility of the peptide sC18-SNBD for fluorescence lifetime imaging microscopy (FLIM) in HeLa cells. Given the fluorogenic character of SNBD, we envisaged that variations in its fluorescence lifetime would enable time-course monitoring of the subcellular localization of the CPP (Figures and). For these experiments, we incubated HeLa cells with sC18-SNBD and acquired real-time fluorescence intensity as well as FLIM images. Importantly, we confirmed that the FLIM readouts of sC18-SNBD were well above the intensity threshold of autofluorescence in unstained HeLa cells (Figure ). Unlike fluorescence intensity readouts (Figure ), the FLIM analysis differentiated three clear lifetime components both in the microscope images (Figure ), histograms (Figure ), and in the phasor plots (Figure ), with the longest lifetimes (~6 ns) corresponding to the initial interactions of sC18-SNBD with the plasma membrane, and shorter lifetimes observed upon internalization to the cytosol and translocation to the nucleus (Figure ). Importantly, the localization of sC18-SNBD in different subcellular compartments provided valuable insights of its mechanisms of internalization. For instance, we observed distinct nuclear localization of sC18-SNBD even at early timepoints (i.e., 10 min), which suggests that this CPP can undergo rapid direct translocation in addition to the reported gradual cellular endocytosis, a dual internalization mechanism that has been reported for some CPPs. 14 Altogether, these results confirm that the environmental sensitivity of SNBD is optimal for FLIM imaging and represents a useful reporter to differentiate the complex pathways of CPP internalization with high spatiotemporal resolution. Fluorescence labeling of chemokine proteins with Pd-SNBD. Next, we decided to expand the scope of our minimal labeling strategy to proteins and labeled the mouse chemokine ligand 2 (mCCL2). mCCL2 is a ~10 kDa small protein that plays a major role in chemotaxis and has been associated with the recruitment of macrophages to the tumor microenvironment. mCCL2 binds to its cognate chemokine receptor CCR2 and enters macrophages via receptor-mediated endocytosis. For our studies, we employed the full-length mCCL2 with two disulfide bonds in the native sequence and an additional Cys residue at the C-terminal end for SNBD labeling (Figure ). First, mCCL2-Cys (50 µM) was incubated with Pd-SNBD (5 equiv.) under non-reducing conditions (20 mM Tris, pH 7.5, 5% DMF, 37°C) for 30 minutes, after which the reaction was quenched with 3-mercaptopropionic acid and purified using a protein desalting column. We analyzed the labeled mCCL2-SNBD by SDS-PAGE electrophoresis, which featured a single band at the correct molecular weight with strong in-gel fluorescence (Figure ). Mass spectrometry analysis further confirmed equimolar labeling with the disulfide bonds of mCCL2-SNBD remaining unaffected (Figure ). Next, we acquired fluorescence microscopy images in live RAW264.7 macrophages as a murine macrophage cell line expressing the chemokine receptor CCR2. We incubated the macrophages with mCCL2-SNBD, and fluorescence images were taken after 1 hour without any washing steps. RAW264.7 cells showed bright fluorescence signals distributed across the cytosol confirming internalization of mCCL2-SNBD (Figure ). Furthermore, we confirmed the receptormediated endocytosis of mCCL2-SNBD by performing the same microscopy experiments in the presence of the CCR2 antagonist RS102895, which led to substantially reduced intracellular fluorescence emission (Figure ). These results confirm that late-stage SNBD labeling is not only compatible with peptide structures but also with full-length proteins, thus opening new opportunities to study the internalization and localization of complex biomolecules (e.g., cytokines) in live cells.