title: 3D high-density localization microscopy using hybrid astigmatic/ biplane imaging and sparse image reconstruction.
abstract: Localization microscopy achieves nanoscale spatial resolution by iterative localization of sparsely activated molecules, which generally leads to a long acquisition time. By implementing advanced algorithms to treat overlapping point spread functions (PSFs), imaging of densely activated molecules can improve the limited temporal resolution, as has been well demonstrated in two-dimensional imaging. However, three-dimensional (3D) localization of high-density data remains challenging since PSFs are far more similar along the axial dimension than the lateral dimensions. Here, we present a new, high-density 3D imaging system and algorithm. The hybrid system is implemented by combining astigmatic and biplane imaging. The proposed 3D reconstruction algorithm is extended from our state-of-the art 2D high-density localization algorithm. Using mutual coherence analysis of model PSFs, we validated that the hybrid system is more suitable than astigmatic or biplane imaging alone for 3D localization of high-density data. The efficacy of the proposed method was confirmed via simulation and real data of microtubules. Furthermore, we also successfully demonstrated fluorescent-protein-based live cell 3D localization microscopy with a temporal resolution of just 3 seconds, capturing fast dynamics of the endoplasmic recticulum.
authors.affiliation: Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
authors.forename: Junhong
authors.initials: J
authors.lastname: Min
authors.affiliation: Institute of the Physics of Biological Systems, École polytechnique Fédérale de Lausanne, Switzerland.
authors.forename: Seamus J
authors.initials: SJ
authors.lastname: Holden
authors.affiliation: Institute of the Physics of Biological Systems, École polytechnique Fédérale de Lausanne, Switzerland.
authors.forename: Lina
authors.initials: L
authors.lastname: Carlini
authors.affiliation: Biomedical Imaging Group, École polytechnique fédérale de Lausanne, Switzerland.
authors.forename: Michael
authors.initials: M
authors.lastname: Unser
authors.affiliation: Institute of the Physics of Biological Systems, École polytechnique Fédérale de Lausanne, Switzerland.
authors.forename: Suliana
authors.initials: S
authors.lastname: Manley
authors.affiliation: Department of Bio and Brain Engineering, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
authors.forename: Jong Chul
authors.initials: JC
authors.lastname: Ye
keywords: (100.6640) Superresolution; (180.6900) Three-dimensional microscopy