(a) Isolated single fluorophore conditions of the coated 96-well plate were controlled using a stepbleaching experiment using the PIF software 1. For stepbleaching analysis, fluorophores were imaged at 5 different positions per well in Tris-HCl pH7.5 buffer (10 kW/cm², 20ms exposure time for 20 sec). Left: Maximum intensity projection of a stack of Alexa 647 fluorophores (AF 647). Top right: representative intensity time trace for one detected fluorophore showing one single bleaching step. Bottom right: stepbleaching distribution for all detected molecules (n = 214). (b) dSTORM acquisition template for organic dyes well plate: a “pumping” phase with 100% of laser power was applied (640 nm: 40 kW/cm², 532 nm: 20 kW/cm² both for 10 sec) in order to excite a maximum of molecules to the triplet state. Immediately after the pumping phase, the single-molecule sequence (“STORM regime”) was automatically launched for a duration of 2 minutes at 50% laser power. For mEOS3.2 isolated proteins, no “pumping” phase was needed and the 405 nm and 561 nm lasers were used at constant power during the acquisition phase. 16 buffers on 3 different fluorophores (48 conditions) were tested.