LAUSR

Abstract Stimulated emission depletion (STED) nanoscopy is a widely used nanoscopy technique. Two‐colour STED imaging in fixed and living cells is standardised today utilising both fluorescent dyes and fluorescent proteins. Solutions to image additional colours have been demonstrated using spectral unmixing, photobleaching steps, or long‐Stokes‐shift dyes. However, these approaches often compromise speed, spatial resolution, and image quality, and increase complexity. Here, we present multicolour STED nanoscopy with far red‐shifted semiconductor CdTe quantum dots (QDs). STED imaging of the QDs is optimized to minimize blinking effects and maximize the number of detected photons. The far‐red and compact emission spectra of the investigated QDs free spectral space for the simultaneous use of fluorescent dyes, enabling straightforward three‐colour STED imaging with a single depletion beam. We use our method to study the internalization of QDs in cells, opening up the way for future super‐resolution studies of particle uptake and internalization.