Three-dimensional biplane spectroscopic single-molecule localization microscopy

Ki Hee Song, Yang Zhang, Gaoxiang Wang, Cheng Sun, Hao F. Zhang*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

23 Scopus citations


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 dedicated 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 hurdles in spectral calibration and nonuniform lateral resolution at different depths. We found that implementing the biplane method based on the already established spatial and spectral imaging channels offers a much more attractive solution for 3D sSMLM. It allows for more efficient use of the limited photon budget and provides homogeneous lateral resolution compared with the 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 Fluor 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 resolution of 50 nm.

Original languageEnglish (US)
Pages (from-to)709-715
Number of pages7
Issue number6
StatePublished - Jun 20 2019

ASJC Scopus subject areas

  • Electronic, Optical and Magnetic Materials
  • Atomic and Molecular Physics, and Optics


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