Lanthanide-doped upconversion nanoparticles (UCNPs) are capable of converting multiple near-infrared (NIR) photons into shorter-wavelength one by utilizing real long-lived, ladder-like energy levels of lanthanide ions. Such unique anti-Stokes emissions are immune to the auto-fluorescence background interference and derive from moderate irradiation compared to conventional multi-photon fluorescence, ideally suitable for imaging applications, coupled with improved imaging depth by NIR excitation light, excellent photostability, multicolor sharp-band emissions and tunable long emission lifetimes. However, the critical concentration quenching on upconversion luminescence generally observed in UCNPs sets barriers to obtain bright high doping UCNPs at low excitation power density, which hampers this promising luminescence probe applying to long-term live cell imaging. We explore the mechanism of Tm³⁺ concentration quenching from ensemble and single-particle levels to design the new generation sub-25-nm ultra-bright highly Tm³⁺-doped UCNPs for high-contrast imaging at limited excitation power density condition. This achievement provides a new strategy for developing small-sized bright highly activator-doped UCNPs and will broaden the applications of UCNPs in microscopic imaging.