Fig. 1

Confocal dSTORM data acquisition process and data simulation. a The pulsed 640 nm excitation light is converted to radial polarisation with a quarter wave plate (QWP) after passing through a single mode fibre (SMF), reflected by a beam splitter (BS) into a galvanometric laser scanner and focused by an oil immersion objective. The collected fluorescent emission from the sample is descanned, passed through the BS, reflected by mirrors (M) and focused onto the pinhole (PH), then onto the single-photon avalanche photodiodes (SPAD) using lenses (L1, L2, L3 and L4). Band pass filters (BP) block scattered excitation light and prevent afterglow effects of the detectors. b The galvo scanner is a one-pixel scanner rastering the image line by line. At time t, only the blue marked part of S is active, representing the horizontal acquisition line of the FLIMbee detector. The acquisition speed in x-direction is sufficiently fast to be neglected during simulations. Only active fluorophores overlapping into the active part of S are rendered (green)