Materials: Lipid solutions in CHCl3 of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dipalmitoyl-snglycero-3-phosphocholine (DPPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), 1,2dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(cap biotinyl) were purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA). Atto647N-labeled 1,2-Dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE-Atto647N) was obtained from ATTO-TEC (Siegen, Germany), Cholesterol (Chol) ≥ 99% from Sigma-Aldrich (St. Louis, MO, USA), Neutravidin from ThermoFisher Scientific (Waltham, MA, USA). Flipper-TR® (Flipper) was purchased from Spirochrome (Switzerland), DiIC18(5) solid (DiD) from Molecular Probes, Life Technologies Corporation (Carlsbad, CA, USA), and Halo-tagged JFX650 from Promega. Deuterated water (D2O), 9,10-Anthracenediyl-bis(methylene)dimalonic acid (ADMA), sodium azide (NaN3), polyvinyl alcohol (PVA, mw = 145 kDa), paraformaldehyde (PFA) and dithiothreitol were obtained from Sigma-Aldrich. Ferrous ammonium sulfate hexahydrate >99% and xylenol orange (XO) sodium salt were purchased from Fischer Scientific (Hampton, NH, USA).

Ferrous Oxidation–Xylenol orange (FOX) assay for detection of lipid hydroperoxides Flipper (2.5 μM) was added to lipid solutions of 50 μg/mL DOPC or DPPC in H2O, incubated in the dark for 15 min and then exposed to 488 nm light illumination for 2, 4 or 6 min (12 mW laser, Cobolt 06mld). FOX solution (final concentrations: 0.25 mM ferrous ammonium sulfate, 25 mM methanolic H2SO4 and 0.1 mM XO in water/methanol 25:75) was added to the lipid suspensions and the absorbance was recorded from 450 nm to 700 nm. Control samples of lipids and Flipper (columns dubbed dark in Figure 1b) were left in the dark for additional 45 min after the 15 min incubation period and then the FOX solution was added. Statistical analysis was performed using GraphPad Prism 10.2.2 (San Diego, CA, USA), P values less than 0.01 were considered statistically significant.

Singlet oxygen (1O2) detection Lipid solutions of DOPC or DPPC at 50 μg/mL in D2O were incubated with Flipper (2.5 μM) for 15 min in the dark. ADMA (6 μM final concentration) was added and the solutions were illuminated for 30 seconds intervals with a 488 nm laser (12 mW, Cobolt 06-mld). The changes in the emission of ADMA were recorded (λex = 375 nm λobs = 390 nm – 500 nm) using a BioTek Synergy H1 microplate reader (Agilent Technologies, Santa Clara, CA, USA). In 1O2 quenching experiments, 10 mM NaN3 was added to the solutions prior to light irradiation. Control dark and light experiments were carried out, where lipid solutions incubated with Flipper were kept in the dark for the duration of the experiments and lipids without Flipper were exposed to the same light dose, respectively.

GUVs formation and imaging GUVs were prepared following the PVA-assisted method with minor modifications.1 Glass coverslips were cleaned by 15 min of sonication in acetone followed by 15 min in alkaline detergent (Hellmanex). Then, 40 μL of PVA solution (5 % w/w) in milliQ water was spread on the coverslip and dried for 30 min at 50 °C. A total volume of 10 µL of the corresponding lipid at 1 mg/mL doped with 0.02 % biotinylated and 0.1 % fluorescently labeled lipids (only in two color experiments) in CHCl3 was spread on the PVA film and placed under vacuum for 1 h. The film was hydrated with 200 μL of 200 mM sucrose solution for 30 min at room temperature for DOPC and POPC, or 48 °C for DPPC and the ternary mixture POPC/DPPC/Chol. GUVs were collected using a cut pipette tip and diluted in 200 mM glucose solution, incubated with 1 µM Flipper (final concentration) for at least 15 min and imaged at room temperature in 18-well glass bottom dishes (Ibidi GmbH, Munich, Germany) previously coated with 50 μg/mL Neutravidin solution for 3 h at 37 °C or overnight at 4 °C.

Confocal and FLIM imaging Confocal and lifetime images were obtained using a TCS SP8 microscope (Leica Microsystems GmbH, Germany) equipped with an HC PL APO CS2 60x/1.40 oil objective, a pulsed white light laser (WLL) operating at 20 MHz repetition rate, 70 % master power and a hybrid detector (HyD) in photoncounting mode and 16-bit depth. Flipper was excited at 488 nm and the emission was collected in the 530-650 nm range, except when imaging vesicles co-labeled with far-red dyes, where detection was narrowed to 530-630 nm. Samples containing Atto647N or DiD were excited at 650 nm and the emission was detected between 670-750 nm. Lifetime measurements and analysis were performed using LAS X software. The lifetimes were calculated by fitting a bi-exponential decay and values from the longest decay were used.2 Averaged lifetime images were obtained using the phasor plots. For phasor analysis, median or wavelet filters with a threshold of 5-8 were applied to better differentiate the photon clouds, which were then manually selected with linear or circular tools of radius 20-25.
Simultaneous FLIM and photo-oxidation experiments were performed at 488 nm by consecutive imaging scans with unidirectional exposure at 400 Hz and using 2x line accumulation, resulting in pixel dwell-times around 4-5 μs and a total time per frame of about 2.5 seconds. The power of the laser was typically set to 25 % of the master power, which resulted in 52 μW maximum power measured at the back of the objective using the bleaching point method provided from Leica, except when other powers were tested as indicated in the text (see Figure S2a). For dual-color imaging, sequential scan mode between lines was used. Cell culture HeLa cells were grown in DMEM medium at 37 °C and 5 % CO2. Transfection of Halo-tagged variants of GPI-AP and TfR was performed using the plasmids Str-KDEL_SBP-Halo-GPI and Str-KDEL_TfR-SBPHalo, respectively, which carry the so-called retention using selective hooks (RUSH) system.3 RUSH is based on the expression of streptavidin (Str) together with a targeting motif (in this case to the endoplasmic reticulum (ER) using the protein KDEL) and the reporter protein (GPI-AP or TfR) linked to a Str-binding peptide (SBP) motif. Once expressed, both proteins are retained in the ER until the addition of 40 μM D-biotin, which induces the displacement of SPB from Streptavidin and the release of the proteins. Cells were incubated at 37 °C for additional 60-90 min after biotin addition to ensure sufficient accumulation of the recombinant proteins at the plasma membrane and then incubated with the far-red Halo-JFX650 (1 μM) for 15 min. Cells were washed three times with warm PBS to remove the unbound dye.

Preparation of GPMVs GPMVs were produced and isolated from HeLa cells as described in Sezgin et al.4 Cells were seeded on 35 mm glass bottom dishes (MatTek Corporation, Ashland, MA, USA) and grown for 24 h to a confluency of around 70 %. To induce the formation of non-phase separated GPMVs, cells were washed twice with GPMV buffer (150 mM NaCl, 10 mM HEPES, 2 mM CaCl2 at pH 7.4) and incubated with 25 mM PFA and 2 mM dithiothreitol for 2 h at 37 °C. Supernatants were collected and centrifuged for 90 seconds at 1000 g to remove detached cells. GPMVs were allowed to settle for 1 h and 20 l from the bottom of the tube were diluted in 130 L of GPMV buffer, incubated with 1 µM Flipper (final concentration) for at least 15 min and transferred to 18-well glass bottom chambers previously coated with 2 mg/ml BSA.