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Two-Dimensional Standing Wave Total Internal Reflection Fluorescence Microscopy: Superresolution Imaging of Single Molecular and Biological Specimens

Euiheon Chung ^*, Daekeun Kim , Yan Cui , Yang-Hyo Kim  and Peter T. C. So  

^* Harvard-Massachusetts Institutes of Technology, Division of Health Sciences and Technology, Departments of Mechanical Engineering, and Biological Engineering, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139; and Department of Physics, Tianjin Polytechnic University, People's Republic of China

Correspondence: Address reprint requests to Peter T. C. So, E-mail: ptso{at}mit.edu.

The development of high resolution, high speed imaging techniques allows the study of dynamical processes in biological systems. Lateral resolution improvement of up to a factor of 2 has been achieved using structured illumination. In a total internal reflection fluorescence microscope, an evanescence excitation field is formed as light is total internally reflected at an interface between a high and a low index medium. The <100 nm penetration depth of evanescence field ensures a thin excitation region resulting in low background fluorescence. We present even higher resolution wide-field biological imaging by use of standing wave total internal reflection fluorescence (SW-TIRF). Evanescent standing wave (SW) illumination is used to generate a sinusoidal high spatial frequency fringe pattern on specimen for lateral resolution enhancement. To prevent thermal drift of the SW, novel detection and estimation of the SW phase with real-time feedback control is devised for the stabilization and control of the fringe phase. SW-TIRF is a wide-field superresolution technique with resolution better than a fifth of emission wavelength or 100 nm lateral resolution. We demonstrate the performance of the SW-TIRF microscopy using one- and two-directional SW illumination with a biological sample of cellular actin cytoskeleton of mouse fibroblast cells as well as single semiconductor nanocrystal molecules. The results confirm the superior resolution of SW-TIRF in addition to the merit of a high signal/background ratio from TIRF microscopy.

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