Plan apochromat 63 1.4 oil dic lens

EGFR-expressing A431 cells were seeded in 24-well cell imaging black plates with glass bottoms (1.5 × 10⁴ cells well⁻¹). After 2 d, the medium was changed, and Alexa Fluor 647-labeled MNT were added to final concentrations of 50 nM.
Prior to imaging, SYBR Green (1:10,000 dilution) was added to the cells to visualize the nuclei. After incubation with Alexa Fluor 647-labeled MNT, the cells were examined under the LSM-510 Meta NLO multiphoton laser scanning microscope fitted with a Plan-Apochromat ×63/1.4 Oil DIC lens (Carl Zeiss, Oberkochen, Germany). SYBR Green fluorescence was recorded at an excitation wavelength of 488 nm and an emission wavelength bandpass of 500 to 530 nm. Alexa Fluor 647 fluorescence was recorded at an excitation wavelength of 633 nm and an emission wavelength bandpass of 650 to 710 nm. The mean intranuclear fluorescence at 7 and at 48 h of Alexa 647-labeled MNT toward SYBR Green were calculated using multiphoton laser scanning microscope software.

Cells were incubated in 0.1% BSA DMEM supplemented with 5 µM laurdan (Molecular Probes, Oregon, USA) in DMSO for 1 h. Cells were then pelleted and resuspended in 250 µL serum-free DMEM before being plated out in 8-well Nunc Lab-Tek II #1.5H glass-bottom chamber wells (Thermo Fisher Scientific, Massachusetts, USA). After allowing the cells to adhere for 20 min at 37 °C, 50 µL of 0.5% BSA solution was added to maintain cell viability during imaging. Lambda stacks were recorded using a Zeiss Elyra PS.1 microscope with a Tokai Hit stage/objective heater attached, and 5% CO₂/humidity maintained (Zeiss, Oberkochen, Germany). An optical zoom of 2 was applied to a Plan-Apochromat 63×/1.4 Oil DIC lens (Zeiss, Oberkochen, Germany) and images were obtained every 8.9 nm from 410 nm to 695 nm using a line average of 16. Recorded stacks were exported to the Spectral Imaging Toolbox in Matlab^{52 (link)}, where the data were segmented to isolate the laurdan signal from the plasma membranes of individual cells and the generalized polarization (GP) of the plasma membrane calculated (Fig. 4a). Images of a reference solution (laurdan in DMSO) were obtained with the same microscope settings as used for the imaging of cells, and a reference value (GP_ref) of 0.207 was used for laurdan^{26 (link)}. For details of GP calculation using the Spectral Imaging Toolbox, see ref. ^{52 (link)}.