title: Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens.
abstract: 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 approximately 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.
authors.affiliation: Harvard-Massachusetts Institutes of Technology, Division of Health Sciences and Technology, Department of Mechanical Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
authors.forename: Euiheon
authors.initials: E
authors.lastname: Chung
authors.forename: Daekeun
authors.initials: D
authors.lastname: Kim
authors.forename: Yan
authors.initials: Y
authors.lastname: Cui
authors.forename: Yang-Hyo
authors.initials: YH
authors.lastname: Kim
authors.forename: Peter T C
authors.initials: PT
authors.lastname: So
mesh_terms: D000199:Actins / Q000737:chemistry; D000818:Animals; D001703:Biophysics / Q000379:methods*; D003599:Cytoskeleton / Q000737:chemistry; D004867:Equipment Design; D005456:Fluorescent Dyes / Q000494:pharmacology; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D008962:Models, Theoretical; D041681:NIH 3T3 Cells; D011137:Polystyrenes / Q000737:chemistry; D045663:Quantum Dots; D012666:Semiconductors; D013997:Time Factors
chemical_list: D000199:Actins; D005456:Fluorescent Dyes; D011137:Polystyrenes