Spectroscopic single-molecule localization microscopy (sSMLM) captures the full emission spectra of individual molecules while simultaneously localizing their spatial locations at a precision greatly exceeding the optical diffraction-limit. To achieve this, sSMLM uses a dispersive optical component to separate the emitted photons into dedicate spatial and spectral imaging channels for simultaneous acquisition. While adding a cylindrical lens in the spatial imaging channel enabled three-dimensional (3D) imaging in sSMLM, the inherent astigmatism leads to technical hurdle in spectral calibration and non-uniform lateral resolution at different depths. We found that implementing biplane method based on the already established spatial and spectral imaging channels offers a much more attractive solution for 3D sSMLM. It allows more efficient utilization of the limited photon budget and provides homogeneous lateral resolution as compared with astigmatism-based method using a cylindrical lens. Here, we report 3D biplane sSMLM and demonstrate its multi-color 3D imaging capability by imaging microtubules and mitochondria in fixed COS-7 cells immunostained with Alexa Flour 647 and CF 660C dyes, respectively. We showed a lateral localization precision of 20 nm at an average photon count of 550, a spectral precision of 4 nm at an average photon count of 1250, and an axial localization precision of 50 nm.
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