Song Ki-Hee, Zhang Yang, Wang Gaoxiang, Sun Cheng, Zhang Hao F
Department of Biomedical Engineering, Northwestern University, Evanston, Illinois 60201, USA.
Department of Hematology, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, Hubei 430030, China.
Optica. 2019 Jun 20;6(6):709-715. doi: 10.1364/optica.6.000709. Epub 2019 May 23.
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.
光谱单分子定位显微镜（sSMLM）可捕获单个分子的完整发射光谱，同时以远超光学衍射极限的精度确定其空间位置。为此，sSMLM使用色散光学元件将发射的光子分离到专用的空间和光谱成像通道进行同步采集。虽然在空间成像通道中添加柱面透镜可实现sSMLM中的三维（3D）成像，但固有的像散会导致光谱校准方面的技术障碍以及不同深度处横向分辨率的不均匀性。我们发现，基于已建立的空间和光谱成像通道实施双平面方法为3D sSMLM提供了一种更具吸引力的解决方案。与使用柱面透镜的基于像散的方法相比，它能更有效地利用有限的光子预算，并提供均匀的横向分辨率。在此，我们报告了3D双平面sSMLM，并通过分别对用Alexa Fluor 647和CF 660C染料免疫染色的固定COS-7细胞中的微管和线粒体进行成像，展示了其多色3D成像能力。我们在平均光子计数为550时展示了20 nm的横向定位精度，在平均光子计数为1250时展示了4 nm的光谱精度，以及50 nm的轴向定位分辨率。
