Laurdan-based Membrane Fluidity Imaging

Late stationary and mid-log phase cultures were normalized to an OD₆₀₀ of 0.7 in PBS and incubated with 100 μM Laurdan for 10 min at 37°C. Cells were washed twice with PBS and 10 μL of the cell suspension was spotted onto PBS-agarose pads (1% wt/vol) mounted on glass slides. Coverslips were placed over the agarose pads and sealed using paraffin wax.
Slides were imaged using a Zeiss LSM 880 laser scanning microscope with Airyscan, using a Plan-Apochromat 63x/1.4 Oil DIC objective with an incubation chamber set to 37°C. The slides were equilibrated for 10 min within the chamber before imaging and excited using a 405-nm laser with emission collected between 419 and 455 nm (blue) and between 480 and 520 nm (green) simultaneously. Digital images were acquired using the Zen (Zeiss) software and analyzed using ImageJ. Using ImageJ, regions of interests (ROIs) of individual cells or cell clusters were selected and mean fluorescence intensities (MFIs) of each ROI for each channel were measured and tabulated in Microsoft Excel. Using the following formula, the average GP values for each ROI were calculated and then plotted using GraphPad Prism software:
$$\mathit{GP}=\frac{I_{\mathit{Blue}}-I_{\mathit{Green}}}{I_{\mathit{Blue}}+I_{\mathit{Green}}}=\frac{I_{419-455\mathit{nm}}-I_{480-520\mathit{nm}}}{I_{419-455\mathit{nm}}+I_{480-520\mathit{nm}}}$$
Laurdan was validated to be responsive to changes in membrane fluidity via control experiments subjecting stained cells to a gradient of temperatures, and membrane fluidizer, benzyl alcohol (Fig. S7B and C).