Laurdan was dissolved in DMSO at a concentration of 1 mM as a stock solution. To measure the steadystate Laurdan uorescence spectrum in cell membrane, HepG-2 and NP-8 cells were seeded in 6-well plates. After 24 h treatments with different liposomes or free 2-OHOA ( rstly dissolved in DMSO at a concentration of 20 mM as stock solution, then diluted in cell culture media for cell treatment), the culture media was carefully removed, and the cells were gently washed with D-PBS. Subsequently, fresh preheated media containing 10 µM Laurdan was introduced into the wells and incubated for 30 minutes in a cell culture incubator shielded from light. Following Laurdan staining, cells were washed and detached using trypsin. The collected cells were suspended in D-PBS and analyzed using a uorescence spectrometer (FP-8500, Jasco, Japan). Steady-state Laurdan spectra were obtained with an excitation wavelength of 345 nm, and emission was collected in the range of 400-600 nm.
The Laurdan steady-state uorescence spectra data from uorescence spectrometer were collected and analyzed. The value (GP value calculated according to steady-state Laurdan spectra) was calculated according to the Eq. ( 2):
2
Where and represent the uorescence intensity at 440 and 490 nm, respectively.
For two-photon microscopy observations, cells were initially cultured in 35 mm Φ glass-bottom dishes.
Laurdan staining was performed as previously described. Following staining, the samples were observed under a two-photon microscopy. To maintain the temperature and CO 2 concentration of the cell samples during microscopy observation, the glass bottom dishes were placed in a living cell imaging chamber equipped with a stage-top incubator (INUB-PPZI, Tokai Hit, Japan) to sustain a 37℃ and 5% CO 2 environment. Two-photon uorescence images of the Laurdan-labeled cells were obtained with an inverted microscopes (Eclipse TE2000-U, Nikon, Japan) with a ×60 water-immersion objective (Plan Apo VC 60×, NA = 1.2, Nikon, Japan). A Ti-sapphire laser (Chameleon Vision II, Coherent, USA) with a repetition rate of 80 MHz and pulse width of 140 femtosecond (fs) was used as the excitation laser. The wavelength peak was tuned to 780 nm and the power was adjusted to 100 mW. The group delay dispersion (GDD) was adjusted to 14,000 femtosecond squared (fs 2 ). Laurdan emission from the cell samples were ltered through 436/20 nm (blue) and 495/25 nm (cyan) bandpass lters. The uorescence intensity of two channels were detected using a laser-scanning uorescence detector (D-Eclipse C1, Nikon, Japan). The relative sensitivities of the two channels were determined using 0.1 mM Laurdan in DMSO, and the calibration factor (G-factor) was calculated (refer to supplementary information). Two-photon microscopy images of Laurdan-stained cell membrane were analyzed using the imageJ software. Laurdan GP images were acquired by calculating the GP value of each pixel. The (GP value calculated according to Laurdan two-photon microscopy images) of each pixel, was calculated according Eq. ( 3).
The Laurdan steady-state uorescence spectra data from uorescence spectrometer were collected and analyzed. The value (GP value calculated according to steady-state Laurdan spectra) was calculated according to the Eq. ( 2):
2
Where and represent the uorescence intensity at 440 and 490 nm, respectively.
For two-photon microscopy observations, cells were initially cultured in 35 mm Φ glass-bottom dishes.
Laurdan staining was performed as previously described. Following staining, the samples were observed under a two-photon microscopy. To maintain the temperature and CO 2 concentration of the cell samples during microscopy observation, the glass bottom dishes were placed in a living cell imaging chamber equipped with a stage-top incubator (INUB-PPZI, Tokai Hit, Japan) to sustain a 37℃ and 5% CO 2 environment. Two-photon uorescence images of the Laurdan-labeled cells were obtained with an inverted microscopes (Eclipse TE2000-U, Nikon, Japan) with a ×60 water-immersion objective (Plan Apo VC 60×, NA = 1.2, Nikon, Japan). A Ti-sapphire laser (Chameleon Vision II, Coherent, USA) with a repetition rate of 80 MHz and pulse width of 140 femtosecond (fs) was used as the excitation laser. The wavelength peak was tuned to 780 nm and the power was adjusted to 100 mW. The group delay dispersion (GDD) was adjusted to 14,000 femtosecond squared (fs 2 ). Laurdan emission from the cell samples were ltered through 436/20 nm (blue) and 495/25 nm (cyan) bandpass lters. The uorescence intensity of two channels were detected using a laser-scanning uorescence detector (D-Eclipse C1, Nikon, Japan). The relative sensitivities of the two channels were determined using 0.1 mM Laurdan in DMSO, and the calibration factor (G-factor) was calculated (refer to supplementary information). Two-photon microscopy images of Laurdan-stained cell membrane were analyzed using the imageJ software. Laurdan GP images were acquired by calculating the GP value of each pixel. The (GP value calculated according to Laurdan two-photon microscopy images) of each pixel, was calculated according Eq. ( 3).
