Tsc sp5 2

Filled DropSOAC capsules were placed in an environmental chamber (Pathology Devices Inc., LiveCell) at 37°C and 85% RH on an inverted confocal microscope (Leica TSC SP5 II). Drops were imaged with a 20× objective in a 60 μm z-stack with 2 μm increments. The middle of the z-stack was set to the center of the drops. Images were taken using brightfield and fluorescence imaging every 30 min for the duration of 21.5 or 4.5 h for the bacterial incubation studies and the drop stability studies, respectively. Z-stacks were used to capture the entire depth of the drop and to ensure that changes in focal plane from temperature fluctuations were captured. Multiple positions were captured for each study using the “Mark and Find” application within the Leica microscopy software (LAS AF). In the water soaking study with ROX dye, a 63X water immersion objective was used.

GUVs were diluted 10-fold in 400 mM glucose solution and added to a BSA-coated observation chamber. Lifetime images were obtained using a Leica TSC SP5 II inverted confocal microscope and a 20x air objective. A Ti:Sapphire laser (Coherent, Chameleon Vision II, 80 MHz) provided two-photon excitation at either 930 nm (premixed vesicles) or at 900 nm (for the in situ oxidation experiments) and fluorescence emission was collected either between 500–580 nm (premixed vesicles) or between 500 and 600 nm (for in situ oxidation). FLIM images (256 × 256 pixels, 256 channels) were acquired using a TCSPC card (Becker & Hickl GmbH®, SPC-830). Instrument response function (IRF) was measured using the second harmonic generation signal from urea crystals. The lifetimes were calculated by fitting the decays to either a monoexponential (BC10, minimum 200 counts per pixel at peak after binning) or biexponential (BC6 + + , minimum 500 counts per pixel at peak after binning) models.