Supplementary Figure 1. Thin layer chromatography of R18 salts with different counterions. The mobility of the R18 salts with TPB counterions is much

Transcription

1 Supplementary Figure 1. Thin layer chromatography of R18 salts with different counterions. The mobility of the R18 salts with TPB counterions is much higher with perchlorate, showing their much higher hydrophobicity. Within TPB counterions the mobility increases with the fluorination level. Eluent: Dichloromethane/methanol, 98/2, TLC plate illuminated at 365 nm.

2 Supplementary Figure 2. Combined AFM and fluorescence imaging of dye-doped PLGA NPs. AFM (a) and fluorescence microscopy (b) images of the same field of NPs loaded with 1 wt% R18/F5-TPB deposited on PEI-covered glass. The exposure time in the fluorescence image was 100 ms. Scale bar, 1 µm.

3 in methanol in buffer Quantum yield ClO4 TPB F1-TPB F5-TPB Dye content (wt%) Supplementary Figure 3. Fluorescence quantum yields vs dye concentration for NPs loaded with R18 salts with different counterions. The quantum yields of the same dyes in methanol and in aqueous buffer are given for comparison ( ex 530 nm). The dotted lines are given to guide the eyes.

4 Fluorescence intensity (a. u.) polymer: PCL PMMA PS PLGA Dye content (wt%) Supplementary Figure 4. Fluorescence brightness of dye-doped NPs from different polymers. Fluorescence intensities ( ex 530 nm) vs R18/F5-TPB concentration inside polycaprolactone (PCL, M n ), poly(methyl methacrylate) (PMMA, M w ), polystyrene (PS, M w ), and PLGA NPs. The size of the NPs was 130, 110, and 115 nm for PCL, PMMA, and PS, respectively. Preparation of PCL and PMMA NPs was analogous to that of PLGA NPs. PS NPs were made by adding a dioxane solution of PS and the dye into water containing 1 mm Tween-80.

5 Fluorescence intensity (a. u.) counterion: ClO4 ClO4 + 1 eq. C 6 F 6 ClO4 + 4 eq. C 6 F 6 F5-TPB Dye content (wt%) Supplementary Figure 5. Effect of hexafluorobenzene on the fluorescence intensity of dye-doped NPs. Fluorescence intensities ( ex 530 nm) vs dye salt concentration inside NPs for R18 perchlorate (ClO4) with addition of 1 and 4 mole eq. of hexafluorobenzene (C 6 F 6 ). The data are compared to the control NPs with encapsulated R18 perchlorate or R18/F5-TPB.

6 Absorbance ClO4 NPs 5 wt% TPB NPs 5 wt% F1-TPB NPs 5 wt% F5-TPB NPs 5 wt% F5-TPB in methanol (nm) Supplementary Figure 6. Effect of the counterion on the absorption spectra of dyedoped NPs. Absorption spectra of NPs containing 5 wt% of R18 salts with different counterions and comparison to R18/F5-TPB in methanol (spectra are normalized to the F5- TPB 5 wt% NPs spectrum).

7 Absorbance F5-TPB NPs 0.1 wt% F5-TPB NPs 0.5 wt% F5-TPB NPs 1 wt% F5-TPB NPs 5 wt% (nm) Supplementary Figure 7. Effect of the dye loading on the absorption spectra of dyedoped NPs. Absorption spectra of F5-TPB NPs containing different wt% of the R18/F5-TPB salt (spectra are normalized to the F5-TPB 5 wt% NPs spectrum).

8 Absorbance ClO4 in water TPB in water F1-TPB in water F5-TPB in water F5-TPB in methanol (nm) Supplementary Figure 8. Absorption spectra of R18 salts in solvents. Absorption spectra of R18 salts with different counterions in water and comparison to R18/F5-TPB in methanol (spectra are normalized to the R18/F5-TPB spectrum in methanol).

9 Fluorescence (a.u.) F5-TPB in methanol ClO4 NPs 5 wt% TPB NPs 5 wt% F1-TPB NPs 5 wt% F5-TPB NPs 5 wt% (nm) Supplementary Figure 9. Effect of the counterion on the fluorescence spectra of dyedoped NPs. Comparison of the normalized fluorescence emission spectra of NPs containing 5 wt% of R18 salts with different counterions and to R18/F5-TPB in methanol.

10 Fluorescence (a.u.) in methanol NPs 0.1 wt% NPs 1 wt% NPs 5 wt% (nm) Supplementary Figure 10. Effect of the dye loading on the fluorescence spectra of dyedoped NPs. Comparison of the normalized fluorescence emission spectra of NPs containing the R18/F5-TPB salt at different concentrations and of R18/F5-TPB in methanol.

11 Absorbance or fluorescence F5-TPB NPs 0.1 wt% abs F5-TPB NPs 0.1 wt% ex F5-TPB NPs 5 wt% abs F5-TPB NPs 5 wt% ex F5-TPB in methanol ex (nm) Supplementary Figure 11. Absorption and fluorescence excitation spectra of different dye-doped NPs. Absorption and fluorescence excitation spectra ( em 580 nm) of R18/F5-TPB NPs containing different wt% of dye and comparison to the excitation spectra of R18/F5-TPB in methanol (spectra are normalized to the F5-TPB 5 wt% NP spectrum).

12 Normalized emission Normalized emission a F5-TPB 0.1 wt% 1 wt% 5 wt% 0.01 b 1E F5-TPB 1 wt% F1-TPB 1 wt% 5 wt% TPB 1 wt% 5 wt% E Time (ns) Supplementary Figure 12. Fluorescence lifetime measurements of dye-doped NPs. Fluorescence emission decays of dye-doped NPs. (a) R18/F5-TPB salt at different wt%. (b) Comparison of F5-TPB salt at 1 wt% loading with F1-TPB and TPB salts at 1 and 5 wt% loading.

13 Longest lifetime component (ns) Contribution to emission (%) 5 longest lifetime component (ns) contribution to emission (%) TPB, 0.1% TPB, 1% TPB, 5 % F1-TPB F1-TPB, 1% F1-TPB, 5% F5-TPB F5-TPB, 1% F5-TPB, 5% Supplementary Figure 13. Values and relative contribution to the emission of the longest lifetime component for various dye-doped NPs. The relative contribution, f, was calculated from the value ( 1 ) and amplitude ( 1 ) of the long-lived lifetime given in Supplementary Table 2, by f = 1 1 / mean.

14 Supplementary Figure 14. Time-resolved anisotropy decays of various dye-doped NPs. The steady-state anisotropy ( ) and residual anisotropy (r ) are given in the inset. For 0.1 wt% F5-TPB, 1 wt% F1-TPB, and 1 wt% TPB the corresponding fits are given, and the residual anisotropy corresponds to the fitted value. For 1 and 5 wt% F5-TPB the lines are guides to the eye. In these cases the residual anisotropies were determined as mean value of the anisotropy between 1 and 4 ns and the given error is the s.e.m.

15 Number of detected particles Time (s) Supplementary Figure 15. Recovery of the single-particle fluorescence after the initial turn off. Number of detected NPs loaded with 5 wt% R18/F5-TPB depending on the start time of detection (black) and on the time over which the detection was performed (red). For the detection of the particles, microscope images of one field (80 µm x 80 µm) obtained with an illumination at 532 nm in the TIRF mode at a power of 4.5 Wcm -2 were analyzed using the particle number function of the ImageJ software. The black symbols show the number of particles detected in one image recorded at time t 1. The red symbols show the number of particles that were detected between time t 1 and time t 2, that is the number of particles that were in the on-state at least once between t 1 and t 2.

16 Frequency W/cm² on off 4.5 W/cm² on off On-/off time (s) Supplementary Figure 16. Frequency of the occurrence of on- and off-times in NPs loaded with 1 wt% R18/F5-TPB at two different illumination powers. Lines represent the corresponding power law fits. Power law exponents were and for on and off-states at an illumination power of 0.45 W/cm², respectively, and and for on and offstates at a power of 4.5 W/cm²; respectively. On- and off-times were determined using a threshold analysis as described in the Materials and Methods section.

17 Supplementary Figure 17. Single-particle emission transients for some dye-doped NPs. Emission transients of single NPs, with 32-ms time resolution for NPS loaded with (a) 1 wt% of R18/TPB and (b) 1 wt% of R18/F1-TPB.

18 Supplementary Figure 18. Co-localization of dye-doped NPs with a lysosome marker in living cells. Micrographs of HeLa cells cultured for 3h with 1 wt% R18/F5-TPB NPs and for 30 min with LysoTracker Green DND-26. The micrographs were obtained using a confocal microscope with excitation at 532 nm (a) and 488 nm (b) to image NPs and LysoTracker, respectively. Scale bar, 10 µm.

19 Supplementary Figure 19. Living cells after incubation with dye-doped NPs at 4 C. Micrograph of HeLa cells cultured for 3h with 1 wt% R18/F5-TPB NPs at 4 C and labelled with WGA-AlexaFluoro 488. The micrograph was obtained using a confocal microscope with excitation at 532 nm (red) and 488 nm (green) to image NPs and cell membranes, respectively. Scale bar, 10 µm. Practically no NPs were observed inside the cells after incubation in these conditions, which strongly slows down endocytosis.

20 Supplementary Figure 20. Living cells after incubation with dye-doped NPs for 7 days. Micrograph of HeLa cells cultured for 7 days with 1 wt% R18/F5-TPB NPs. The micrograph was obtained using a confocal microscope with excitation at 532 nm. Scale bar, 20 µm. The NPs are clearly visible inside the cells, though their concentration has decreased mainly due to cell division.

21 Viability wt% 1 wt% 5 wt% E-4 7.5E NP concentration (g/l) Supplementary Figure 21. Cytotoxicity of dye-doped NPs. Viability of HeLa cells exposed for 72 h to different concentrations of PLGA NPs loaded with 0, 1, and 5 wt% R18/F5-TPB. The highest concentration of the 5 wt% NPs corresponds to a 10 µm concentration of the dye, that is >500-fold larger than the highest concentration used for cellular imaging. Cell viability was tested using MTT. The presence of serum within the first 3 h of incubation did not have any influence on the cytotoxicity (results shown were obtained in the presence of serum).

22 Supplementary Table 1. Dynamic light scattering data (mean size based on volume statistics, polydispersity and zeta potential) for NPs loaded with different amounts of R18 salts. dye salt loading (wt%) diameter (nm) polydispersity (PDI) zeta potential (mv) (±3) (±1) R18/ClO4 a (±6) (±2) (±5) (±6) (±5) (±50) (±400) R18/TPB (±6) (±1) (±5) (±3) (±3) 2 40 (±5) (±400) R18/F1-TPB (±2) (±4) (±3) (±6) (±5) 2 39 (±10) (±15) (±3) R18/F5-TPB (±6) (±4) (±4) (±2) (±3) 2 39 (±3) (±8) (±2) a For the perchlorate counterion, the zeta potential measurements were not possible due to sample aggregation at dye loadings >1 wt%.

23 Supplementary Table 2. Time-resolved fluorescence parameters of NPs loaded with different amounts of R18 salts. a dye salt loading mean wt% (ns) (ns) % (ns) % (ns) % R18/ClO4 in methanol R18/ClO R18/TPB R18/F1-TPB R18/F5-TPB a The values of the individual lifetimes i as well as their amplitudes i are reported. The decay parameters of R18 in methanol are given for comparison. The mean lifetime is given by: mean = i i /100.

24 Supplementary Table 3. Fluorescence anisotropy data of NPs loaded with different amounts of R18 salts. dye salt loading wt% steady state anisotropy R18/TPB R18/TPB R18/TPB R18/F1-TPB R18/F1-TPB R18/F1-TPB R18/F5-TPB R18/F5-TPB R18/F5-TPB