3.5. Bilayer and cell membrane dc-FCCS measurements

dc-FCCS and single-channel fluorescence correlation spectroscopy (sc-FCS) measurements were carried out on a Zeiss Laser Scanning Microscope 780 (Zeiss LSM 780) equipped with a Confocor3 module. CW 488-nm argon-ion and/or 561-nm DPSS lasers were used to excite mEGFP and/or mCherry, respectively, via a 100×, NA 1.4, oil-immersion, plan-apochromat Zeiss objective lens. The fluorescence emission from the focal volume was collected through the same objective lens and directed to the PMTs via a dichroic beam splitter (MBS488 for green only; or MBS488/561 for red-only or for dual channel), tunable spectral filters with transmission in 499-553 nm and 606-695 nm spectral windows for the green and red channels respectively, and a pinhole. The pinhole diameter was set at 1 AU ( = 79 to 80 µm), and its lateral position was optimized daily using calibration fluorescent dyes. The laser powers were set at 0.1 and 0.05 for 488 and 561 lasers, respectively, on the software, which resulted in 180 nW and 800 nW at the objective and gave rise to counts per molecule of 150-350 Hz in the green channel and 250-400 Hz in the red channel for mEGFP and mCherry, respectively. At this excitation setting, both mEGFP and mCherry were operating well below fluorescence saturation (Fig. S2). In comparison, the background count rates were 20-30 Hz in the green and 50-60 Hz in the red channels. Default settings were used for all other acquisition parameters. For dc-FCCS measurements on SLB, focusing was performed by maximizing the photon counts while adjusting the z-position. For dc-FCCS measurements on cells, the basal membrane of the cell was brought into focus in the confocal imaging mode. Measurements were acquired in sets of 15-second runs and saved as *.fcs files. Definite focus functionality of the Zeiss LSM 780 microscope ensured that any axial drift was actively corrected. However, the runs containing discernable changes in the fluorescence intensity due to active drift correction were discarded. Equilibration of the microscope at 37°C (SLB) or 25°C (cells) for approximately 1-2 hours ensured minimal drift. Background fluorescence was acquired with MilliQ water as the sample with the focus a few microns above the coverslip-water interface.