**4. Conclusions**

A novel fluorescent molecule was designed, synthesized, and optically characterized as a probe for lipid membranes. From the optical and confocal microscopy analysis of GUVs loaded with the probe, the probe is able to anchor to the lipid double layer, without internalizing. In addition, the fluorescence of the 3HF18 probe is visible in both Ld and Lo vesicles. MD simulations show that the insertion of the probe does not disturb the membrane, since both the membrane thickness and the order parameter remain essentially unchanged. Furthermore, thanks to a new semi-semantic approach, it was possible to assess that Ld membranes are more sensitive to probe insertion, and that the 3HF18 molecule shows a preference for the Lo or Ld phases of a model membrane, depending on the most stable tautomeric form.

**Supplementary Materials:** The following are available online, Table S1: Thickness values (in Å) of the two membranes with and without the probe, Figure S1: SCD of the unsaturated POPC chain with (blue) and without (green) the probe, Figure S2: SCD of POPC saturated chain with (blue) and without (green) probe, Figure S3: SCD of the DPPC Sn-1 palmitoyl chain (DPPC-Chol) with and without probe, Figure S4: SCD of the DPPC Sn-2 palmitoyl chain (DPPC-Chol) with and without probe, Figure S5: Fluorescence spectra of probe 3HF18 in binary mixture MeOH/H2O in different ratios, Table S2: Results of t-Student test.

**Author Contributions:** Conceptualization, S.C. and S.P.; methodology, L.S. and F.R.; investigation, M.D.M., B.P., A.M.N. and Y.M.; data curation, L.S. and A.M.N.; writing—original draft preparation, M.D.M.; writing—review and editing, S.C. and S.P.; supervision, F.R. and B.P.; funding acquisition, S.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by the Italian Ministry of Education, University and Research MIUR (grant number 300395FRB18), and by the project Clinglio, within the European Commission H2020 Framework Programme (agreement number 755179).

**Acknowledgments:** Jlenia Brunetti is gratefully acknowledged for data acquisition at the confocal microscope.

**Conflicts of Interest:** The authors declare no conflict of interest.
