Interaction of PDI and VWF shown by 2-color STORM super-resolution microscopy. Endogenous VWF and PDI in HUVECs were detected by indirect immunofluorescence employing rabbit anti-VWF plus goat anti-rabbit Alexa Fluor 488 (shown in green) and mouse anti-PDI plus goat anti-mouse Alexa Fluor 647 (shown in red), respectively. Colocalization is shown in yellow. Locations of magnified ROIs in the cell (left) are indicated by white squares and numbers. STORM data sets were acquired on a Nikon N-STORM microscope with an Apo TIRF 100× oil immersion objective (NA 1.49), a back-illuminated EMCCD camera, and a quadband filter composed of a quadline beam splitter and a quadline emission filter. For excitation of Alexa Fluor 647 and 488, a 647-nm continuous wave fiber laser, and the 488-nm line of an argon gas laser were used. For multicolor imaging, the lasers were switched on and off alternately, controlled by an acousto-optic tunable filter. The integration time of the EMCCD camera was set to 16 ms per frame, with an EM gain of 300. Super-resolution images were reconstructed from a series of 12 500 images per channel, using the N-STORM analysis module v. 3.3.1.15801 of NIS Elements AR v. 4.20 with overlapping peak detection enabled. Scale bar in the left cell image represents 5 µm; in magnified images, 100 nm.