title: Shadowless-illuminated variable-angle TIRF (siva-TIRF) microscopy for the observation of spatial-temporal dynamics in live cells.
abstract: Total-internal-reflection fluorescence (TIRF) microscopy provides high optical-sectioning capability and a good signal-contrast ratio for structures near the surfaces of cells. In recent years, several improvements have been developed, such as variable-angle TIRF (VA-TIRF) and spinning TIRF (sp-TIRF), which permit quantitative image analysis and address non-uniform scattering fringes, respectively. Here, we present a dual-color DMD-based shadowless-illuminated variable-angle TIRF (siva-TIRF) system that provides a uniform illumination field. By adjusting the incidence angle of the illuminating laser on the back focal plane (BFP) of the objective, we can rapidly illuminate biological samples in layers of various thicknesses in TIRF or hollow-cone epi-fluorescence mode. Compared with other methods of accomplishing VA-TIRF/sp-TIRF illumination, our system is simple to build and cost-effective, and it provides optimal multi-plane dual-color images. By showing spatiotemporal correlated movement of clathrin-coated structures with microtubule filaments from various layers of live cells, we demonstrate that cortical microtubules are important spatial regulators of clathrin-coated structures. Moreover, our system can be used to prove superb axial information of three-dimensional movement of structures near the plasma membrane within live cells.
authors.affiliation: Department of Cognitive Sciences, Institute of Basic Medical Sciences, Beijing 100850, China ; The State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardio Metabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing 100871, China.
authors.forename: Weijian
authors.initials: W
authors.lastname: Zong
authors.affiliation: The State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardio Metabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing 100871, China.
authors.forename: Xiaoshuai
authors.initials: X
authors.lastname: Huang
authors.affiliation: The State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardio Metabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing 100871, China.
authors.forename: Chi
authors.initials: C
authors.lastname: Zhang
authors.affiliation: The State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardio Metabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing 100871, China.
authors.forename: Tianyi
authors.initials: T
authors.lastname: Yuan
authors.affiliation: Department of Cognitive Sciences, Institute of Basic Medical Sciences, Beijing 100850, China.
authors.forename: Ling-Ling
authors.initials: LL
authors.lastname: Zhu
authors.affiliation: Department of Cognitive Sciences, Institute of Basic Medical Sciences, Beijing 100850, China ; fanmingchina@126.com.
authors.forename: Ming
authors.initials: M
authors.lastname: Fan
authors.affiliation: The State Key Laboratory of Biomembrane and Membrane Biotechnology, Beijing Key Laboratory of Cardio Metabolic Molecular Medicine, Institute of Molecular Medicine, Peking University, Beijing 100871, China.
authors.forename: Liangyi
authors.initials: L
authors.lastname: Chen
keywords: (050.0050) Diffraction and gratings; (110.0110) Imaging systems; (180.0180) Microscopy; (260.6970) Total internal reflection