illustrates schematically the investigated Bioshuttle-conjugates.

displays the dependency of different FFM parameters on the measuring depth with Atto565 [20 mM]. The calculated particle number (black) and count rate (blue) feature a near identical run of the curves approximating the glass surface whereas the diffusion time (red) remains relatively constant. The graph of the averaged ACF (brown /×) shows a higher diffusion time slightly increasing near the glass surface.

the confocal FFM micrographs exhibit the intracellular distribution of CPP-Rd110 A in HeLa cells (picture α) and in MCF7 cells (picture β) 2.5 h after application. The red fluorescence signals are located almost exclusively in vesicle like structures inside of the cytoplasm.

shows the absorption spectra of the cells's supernatants 1h after incubation with (Atto647N)-CPP-S^∩S-NLS-(Atto488)-BioShuttle B. The curves are colour-coded as follows: initial concentration (black), probe 1 (blue), probe 2 (dark blue), supernatant of the Hela S3 probe 1 (brown), supernatant of the Hela S3 probe 2 (red).

shows confocal FFM images of HeLa (left), MCF7 (middle), and TP366 (right) cell lines 24 h after incubation with CtsB-PNA-BioShuttle C. All investigated cell lines show the successful uptake of CtsB-BioShuttle C as shown by the cytoplasm located fluorescence signals. In HeLa cells (left picture) the fluorescence signal is identified perinuclearly inside of the cell; however, no signal is shown inside of the nucleus. The middle picture illustrates MCF7 cells whose nuclei show band like fluorescence signals near the nuclear envelope and inside of the nuclei. TP366 glioma cells (right picture) show band like fluorescence signals inside of the cell nucleus.

a) represents the auto-correlation functions (ACFs) of the performed Alexa Fluor® 488 measurements (n = 60). b) illustrates the decline of the autocorrelations under increasing concentrations of Alexa Fluor® 488 dye. Colour-coded curves of the dilution series: 0.2 nM (blue), 2 nM (red), 10 nM (yellow), 20 nM (magenta), 50 nM (black), 100 nM (brown), 200 nM (green). c) shows the graphs of the diffusion coefficients (ordinate) and the temperature profile (abscissa). , , are equivalent to one and four runs and represents reference values. d) shows the plots of the corresponding diffusion times [µs] (red ordinate) and the focus amplitude (blue ordinate), the abscissa represents the laser output [µW]. The standard deviations of the diffusion times decreased () when the laser output increased, and in parallel the focus amplitude (excitation volume) increased ()] (n = 6).

the graphs display the dependency of different FFM parameters on the measuring depth with CPP-Rd110 A [20 nM] without (A) and with NP40 detergent (B) [0.02 %]. The abscissa represents the spatial distance of the measured molecule from the glass's surface [µm]. Graph A demonstrates diagrams of the particle number (black), the counting rate (blue) [kHz], and the diffusion time (red) [µs] similar to the Atto565 diagrams (Figure 3).

illustrates the Z-scan through a HeLa S3 cell 1 h after incubation with CPP-Rd110 BioShuttle A [200 nM]. The series of pictures show optical longitudinal sections [300 nm] taken from bottom to top. The total high of the stack amounts to 9.3 µm.

The upper part illustrates the identical cross section of pictures at the time points of 30 sec (left) and 60 min (right) of a long-time uptake-experiment during 60 min with (Atto647N)-CPP-S^∩S-NLS-(Atto488)-BioShuttle B into HeLa cells. The pictures represent a superimposition of the red and the green channels. The cells, marked 1 and 2 (left picture), were used for FCS measurements. The zoom into the image reveals particular signals with mixed fluorescence (3× arrows in the right picture). The graphs in the lower part of the figure visualize the intracellular fluorescence intensities relating to the separated transporter (Atto647N)-CPP-S^∩S-NLS-(Atto488) B. The two arrays of curves indicate the intensity of the respective dye. The upper curves represent the fluorescence of Atto488, the lower array curves that of Atto647N in two selected cells during the time series under the Nikon A1Rsi. No saturation of the signal intensity is visible.