*4.3. Confocal Imaging*

Fluorescent proteins associated with the SLB were imaged using a dual-colodr confocal fluorescence fluctuation microscope (Insight, Evotec Technologies, Hamburg, Germany, now Perkin-Elmer, Waltham, MA, USA). Excitation was provided simultaneously at *λ*ex = 488 nm and *λ*ex = 635 nm by two lasers (Sapphire 488-20 CDRH and Radius 635-25, both from Coherent, Santa Clara, CA, USA), whose beams were combined into a single optical fiber, before being focused into the sample using a water immersion objective (U-Apo, 40×, 1.15 NA, Olympus, Tokyo, Japan). The fluorescence signal was refocused through a 40 μm confocal pinhole, separated into green and red detection channels by a dichroic mirror and detected by two avalanche photodiodes (SPCM-CD3017, Perkin-Elmer). Before each experiment, the confocal pinhole was aligned and the observation volume was calibrated in both channels by performing a FCS experiment using nanomolar solutions of Alexa488 (diffusion coefficient *D* = 390 μm<sup>2</sup> s<sup>−</sup><sup>1</sup> at 20 °C [81], *λ*em = 525 nm) and Alexa647 (diffusion coefficient *D* = 290 μm<sup>2</sup> s<sup>−</sup><sup>1</sup> at 20 °C [82], *λ*em = 665 nm), both purchased from Molecular Probes (now Thermo Fisher). Calibration experiments were performed in a sample chamber prepared with a microscope coverslip coated with mica, exactly as the SLB sample to be imaged. Typically, the 1/*e*<sup>2</sup> radius of the detection volume

was estimated to be *wg* = 320 nm in the green detection channel and *wr* = 370 nm in the red detection channel, a bit larger than the expected values in ideal conditions *wg* = 0.51(*<sup>λ</sup>*ex + *<sup>λ</sup>*em)/2/(<sup>2</sup> ln 2)1/2/NA = 190 nm and *wr* = 245 nm. A second calibration step was then carried out using nanomolar solutions of Bax-HyLite488 and cBid-Alexa647, also through a mica-coated coverslip, to determine their molecular brightness. At the 20 μW excitation laser power used in each channel for these experiments, the molecular brightness of Bax-HyLite488 was found to be *Bg* = 6(1) kHz and that of cBid-Alexa647 to be *Br* = 11(2) kHz. Finally, dual-color z-stacks of 10 μm by 10 μm (100 × 100 pixels) images were acquired with an oversampling pixel size *d* = 100 nm and a pixel dwell time *δ* = 1 ms for multiple regions of interest in each sample. Typically, each stack contained 5 or 10 pairs of images, spaced by 0.5 or 1 μm, centred around the focal plane. For each stack, the pair of images with the highest maximum intensity was considered to correspond to the images for which the membrane plane was in focus, and selected for single particle analysis.
