Supporting Matierial for "Generalized Polarization and time-resolved fluorescence provide evidence for different populations of Laurdan in lipid vesicles"

S1 Supporting Information for Generalized Polarization and time-resolved fluorescence provide evidence for different populations of Laurdan in lipid vesicles Mihaela Bacaluma, Mihai Radu a, Silvio Osella b, Stefan Knippenberg c,d and Marcel Ameloot c,* a.Department of Life and Environmental Physics, Horia Hulubei National Institute for Physics and Nuclear Engineering, Reactorului, 30, Măgurele, 077125, Romania. b. Chemical and Biological Systems Simulation Lab, Centre of New Technologies, University of Warsaw, Banacha 2C, 02-097 Warsaw, Poland. c. Biomedical Research Institute, Hasselt University, Agoralaan Bldg. C, 3590 Diepenbeek, Belgium. d.Theory Lab, Hasselt University, Agoralaan Bldg. D, 3590 Diepenbeek, Belgium Results S2 Fluorescence Spectroscopy 400 420 440 460 480 500 520 540 560 580 600 0.0 0.2 0.4 0.6 0.8 1.0 Relative Intensity  / nm 60 oC 15 oCA 400 420 440 460 480 500 520 540 560 580 600 0.0 0.2 0.4 0.6 0.8 1.0 5 oC 60 oC B Relative Intensity  / nm 400 420 440 460 480 500 520 540 560 580 600 0.0 0.2 0.4 0.6 0.8 1.0 C DPPC - 15oC DPPC - 60 oC DMPC - 5 oC DMPC - 60 oC Normalized Intensity  / nm Figure S1. Fluorescence emission spectra of Laurdan inserted into the membrane of LUVs composed of DPPC (A) or DMPC (B) at temperatures ranging from 5 to 60 oC (with a 5 oC step) and (C) Peak normalized emission spectra of Laurdan in DPPC and DMPC. Fluorescence Microscopy S3 Figure S2. Laurdan in GUVs prepared from DPPC (So phaseroom temperature). Representative fluorescence intensity images of Laurdan recorded in the blue channel (A and C) and in the green channel (B and D) with linearly polarized light (first row) and circularly polarized excitation light (second row) for. GP images (E and F) and fluorescence lifetime images (G-J) recorded in the blue channel (G and I) and green channel (H and J). The color code bar in the middle reflects the GP values and the bar on the right indicates the lifetime values in ns. The orientation of the linear polarization of the excitation light is horizontal for the images presented in the top row. The photoselection effect shows up when the light is linearly polarized. The fluorescence lifetimes in the membrane regions oriented parallel to the polarization direction of the incident light are characterized by values lower than the ones recorded from the regions oriented perpendicularly to this direction. In the case of circularly polarized light this distinction is not observed. The images also show a smaller GUV outside and within the GUV. The size of the scale bar (panel J) is 10 µm and applies to all images. Figure S3. Laurdan in GUVs prepared from DOPC (Lα phase – room temperature). Representative fluorescence intensity images of Laurdan recorded in the blue channel (A and C) and in the green channel (B and D) with linearly polarized excitation light (first row) and circularly polarized light (second row). GP images (E and F) and fluorescence lifetime images (G-J) recorded in the blue channel (G and I) and green channel (H and J). The color code bar in the middle reflects the GP S4 values and the bar on the right indicates the lifetime values in ns. The scale of the color bars is the same as that of the corresponding color bar in Fig. S2 for easy comparison. The orientation of the linear polarization of the excitation light is horizontal for the images presented in the top row. No pronounced photoselection effect can be observed in the intensity images. However, in the GP and fluorescence lifetime images, the membrane regions oriented parallel to the polarization direction of the incident light are characterized by values lower than the ones recorded from the regions oriented perpendicularly to this direction. In the case of circularly polarized light this distinction is not observed. The size of the scale bar (panel J) is 10 µm and applies to all images. Figure S4. Microfluorimetric determination of GP of DPPC and DOPC GUVs under circularly polarized two-photon excitation at room temperature. The GP values are averages over 27 GUVs for DPPC and over 15 GUVs for DOPC. The error bars denote the standard deviation. S5 Figure S5. Microfluorimetric determination of lifetimes in blue and green channels for DPPC and DOPC GUVs under circularly polarized two-photon excitation at room temperature. The values are averages over 27 GUVs for DPPC and over 15 GUVs for DOPC. The error bars denote the standard deviation. S6 Figure S6. Variation of the angle of the Laurdan TDM with the z-axis for windows of 20 ns between 240 and 400 ns in both MD runs.