Fig. 3. Transport experiments in cells. (a) Control experiments in HeLa cells showing confocal micrographs of cells incubated with pyranine (5 μM) and cage C (5 μM); nuclei stained with Hoechst (blue). (b) Top: confocal micrographs of HeLa cells incubated with both pyranine (5 μM, green) and ^TmR₄C (5 μM, red) for 30 min in HKR buffer, and washed with HKR buffer before imaging; nuclei stained with Hoechst (blue). The arrow marks the nucleolar accumulation of the peptide–cage carrier. Bottom: confocal images and orthogonal projection of the same HeLa cells showing ^TmR₄C (red) and pyranine (green) cytosolic distribution and partial endosomal co-localization. (c) Transport of pyranine (15 μM, green) in the presence of ^TmR₄C (15 μM), after incubation for 30 min in Vero cells, in HKR. (d) Competition experiments (Vero cells, 30 min of incubation) for ^AcR₄C (10 μM) combined with pyranine (20 μM, green) in the presence of TAMRA (20 μM, red). (e) Transport experiments (HeLa) of pyranine (5 μM) with ^TmR₄C (5 μM) after 30 min, followed by several washes, and subsequent 30 min incubation with 2 μM DAPI to check membrane integrity. (d and e) Red (top) and blue (bottom) fluorescence channels for the TAMRA and DAPI respectively with pyranine (green in both cases). Arrows indicate residual (red (top) and blue (bottom)) fluorescence. Insets show differential interference contrast (DIC) images. Excitation and emission wavelengths: DAPI and Hoechst: λ_exc = 405 nm and λ_em = 450/50 nm; pyranine: λ_exc = 488 nm and λ_em = 525/50 nm; TAMRA: λ_exc = 561 nm and λ_em = 620/60 nm.