Two-dimensional standing wave total internal reflection fluorescence microscopy: superresolution imaging of single molecular and biological specimens

Biophys J. 2007 Sep 1;93(5):1747-57. doi: 10.1529/biophysj.106.097907. Epub 2007 May 4.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Actins / chemistry
  • Animals
  • Biophysics / methods*
  • Cytoskeleton / chemistry
  • Equipment Design
  • Fluorescent Dyes / pharmacology
  • Image Processing, Computer-Assisted
  • Mice
  • Microscopy, Fluorescence / methods*
  • Models, Theoretical
  • NIH 3T3 Cells
  • Polystyrenes / chemistry
  • Quantum Dots
  • Semiconductors
  • Time Factors

Substances

  • Actins
  • Fluorescent Dyes
  • Polystyrenes