Abstract
Semiconductor nanocrystals and their variants are widely used in biological research as fluorescent probes. Their unique characteristics, such as intense brightness, tunable emission properties, and resistance to photobleaching, make them ideal candidates for single-molecule imaging and tracking with localization precision far beyond the diffraction limit. Their fluorescence polarization states and emission spectra can be further utilized to probe changes in their mechanical properties and residing nanoenvironments. We developed a three-dimensional (3D), polarization-sensitive, spectroscopic photon localization microscopy (3D-Polar-SPLM) that enables parallel 3D tracking of individual quantum rods (QRs) while simultaneously capturing their fluorescence spectra and polarization states. Using 3D-Polar-SPLM, we spatially localized individual QRs with a lateral localization precision of 8 nm and an axial localization precision of 35 nm. In addition, we achieved a spectral resolution of 2 nm and a polarization angle measuring precision of 8 degrees. The spectral profile of the fluorescence emission provided a particle-specific signature for identifying individual QRs among the heterogeneous population, which significantly improved the fidelity in parallel 3D tracking of multiple QRs. We envision that this technology will provide new possibilities to reveal the real-time molecular dynamics of biological processes.