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Biomedical Optics Express

Biomedical Optics Express

  • Editor: Joseph A. Izatt
  • Vol. 5, Iss. 5 — May. 1, 2014
  • pp: 1530–1540

Shadowless-illuminated variable-angle TIRF (siva-TIRF) microscopy for the observation of spatial-temporal dynamics in live cells

Weijian Zong, Xiaoshuai Huang, Chi Zhang, Tianyi Yuan, Ling-ling Zhu, Ming Fan, and Liangyi Chen  »View Author Affiliations


Biomedical Optics Express, Vol. 5, Issue 5, pp. 1530-1540 (2014)
http://dx.doi.org/10.1364/BOE.5.001530


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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.

© 2014 Optical Society of America

OCIS Codes
(050.0050) Diffraction and gratings : Diffraction and gratings
(110.0110) Imaging systems : Imaging systems
(180.0180) Microscopy : Microscopy
(260.6970) Physical optics : Total internal reflection

ToC Category:
Microscopy

History
Original Manuscript: February 7, 2014
Revised Manuscript: April 8, 2014
Manuscript Accepted: April 8, 2014
Published: April 15, 2014

Citation
Weijian Zong, Xiaoshuai Huang, Chi Zhang, Tianyi Yuan, Ling-ling Zhu, Ming Fan, and Liangyi Chen, "Shadowless-illuminated variable-angle TIRF (siva-TIRF) microscopy for the observation of spatial-temporal dynamics in live cells," Biomed. Opt. Express 5, 1530-1540 (2014)
http://www.opticsinfobase.org/boe/abstract.cfm?URI=boe-5-5-1530


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