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The spectroscopic information and the corresponding polarization states of a single-molecule emission possess wealth molecule-specific signatures that can be used to reveal the unique molecular electronic state, conformation, and its interactions with the host media. However, existing spectroscopic methods and advanced image analysis techniques, which can potentially provide quantitative analytical tools for the study of cellular dynamics, are yet limited by the diffraction limit. Therefore, developing a nanoscopic imaging platform for simultaneous acquisition of multiple molecular specific properties is highly desirable. Here we report a three-dimensional (3D), polarization-sensitive, spectroscopic photon localization microscopy (3D-Polar-SPLM) that simultaneously captures nanoscopic location of individual fluorescent emitters and their corresponding optical spectra and polarization states. To evaluate the capability of the imaging system, we imaged model system consisting quantum rods (QRs). 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 at a temporal resolution of 10 ms. Except its versatility, 3D-Polar-SPLM further provides advantageous in practical applications since it only employs a single light-path and therefore, is compatible with existing PALM/STORM, potentially bringing immediate impact to the broader research community, across physics, chemistry, material science and biology.
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