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.
Highlights
Various fluorescence-imaging techniques are used to visualize dynamic movements within live cells
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
The authors would like to thank Liqiang Tang and COLD SPRING SCIENCE Corporation for technique support of microscope
Summary
Various fluorescence-imaging techniques are used to visualize dynamic movements within live cells. Organelles within cells exhibit various refraction indices (RIs) that are different from that of the extracellular solution; these structures transiently perturb the TIRF illumination field and introduce interference fringes upon approaching the plasma membrane [6]. To resolve this problem, it is common to rotate the incidence angle of the illuminating laser around the back focal plane (BFP) of the objective using a wedge, a tip/tilt mirror or an acoustic-optical deflection system (AOD) [4, 5, 7]. The TIRF images become homogeneous, and fringes are suppressed
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