Blinking fluorescent probes for tubulin nanoscopy in living and fixed cells

Here we report a small molecule probe for single molecule localisation microscopy (SMLM) of tubulin in living and fixed cells. We explored a series of constructs composed of taxanes and spontaneously blinking far-red dye hydroxymethyl silicon-rhodamine (HMSiR). We found that the linker length profoundly affects the probe permeability and off-targeting. The best performing probe, HMSiR-tubulin, is composed of cabazitaxel and 6-regioisomer of HMSiR bridged by a C6 linker. Microtubule diameters of [≤]50 nm can be routinely measured in SMLM experiments on living and fixed cells. HMSiR-tubulin also performs well in 3D stimulated emission depletion (STED) microscopy, allowing a complementary use of both nanoscopy methods for investigating microtubule functions in living cells.