STORM imaging and analysis

For STORM imaging, µ-Slide 8-well glass bottom chambered coverslips (Ibidi) were cleaned with 1 M NaOH (Sigma) for 1 h, and coated with PLL (0.1%) for 1 h at RT before use. Fixed cells labeled with AF647- or AF568-conjugated antibodies and suspended in 300 μl PBS were placed in each well incubated for 1 h at RT, and then the buffer was exchanged with blinking buffer [PBS containing 0.5 mg mL⁻¹ glucose oxidase, 40 μg mL−¹ catalase, 10% (w/v) glucose, 50 mM cysteamine, and 93 mM Tris-HCl (all from Sigma)]. All buffers were filtered with 0.2 μm-pore filters (Corning) and freshly mixed before use. For fiducials, Fluorescent Nanodiamond (FND) (100 nm, Adamas Nano) diluted in water (1:1000) was sonicated for 15 min and then added to the PLL-coated wells for 30 min. Unbound FNDs were washed 5x with distilled water and then blinking buffer was added. STORM imaging was acquired with a UAPON100XOTIRF1.49NA oil objective (Olympus) at epi-illumination mode on Nanoimager (ONI) microscope implemented with a cylindrical lens for localization in z-dimension. The focus was maintained by the autofocus module of the system. Excitation lasers 532 and 640 nm and the two channels were separated with dichroic (640 LP) and emission (584/80 and 685/40) filters. 532 and 640 nm laser lines were operating at a power of 27.5 and 13.8 kW cm⁻², respectively. For each channel, 30,000 frames (3000 frames x 10 movies) were recorded with a speed of 10 ms per frame. The pixel size on the detector was 117 nm. The two-channel registration with FND fiducial images in the two-channel and single molecule localization processing was performed using Nanoimager software (version 1.1.6165-012f4ed3; ONI). Data analysis and reconstruction of images were performed with custom-written Matlab (MathWorks, 2018a) scripts. Single molecule localization data were thresholded as: 0–14 nm for “precision in x, y (nm)”;−400 to 400 nm for “z range”; 20,000 for “number of photons”; 100 for “background”; 0–2.5 for “Point spread function σX and σY (pixel)'; 0.6–1.5 for “σX/σY” (pixel)'. Channel correction for individual cell images was done with low-resolution images of the single molecules collected in all frames rendered with the original pixel size by calculating the shifted peak of the 2D cross-correlation image obtained using a 2D Gaussian fit. The tip positions were determined using the 2D rendered image (ignoring z localization) as described in the area segmentation section below. Molecules that are localized within the 9 × 9 pixels (corresponding to 1.053 µm × 1.053 µm) around the tip positions were analyzed for the quantification. 3D-pair-correlation (g(r)) between the l-sel and CD45 was calculated. Briefly, the mean count of all CD45 molecules localized within a distance between r (radii) and r + dr (dr = 10 nm) away from each l-sel molecules was calculated, and then it was divided by the volume of the spherical shell. Finally, this number was divided by the total density of the CD45 molecules.