Review

Histochemistry and Cell Biology

, Volume 130, Issue 5, pp 807-817

Open Access This content is freely available online to anyone, anywhere at any time.

Widefield fluorescence microscopy with extended resolution

  • Andreas StemmerAffiliated withNanotechnology Group, Department of Mechanical and Process Engineering, ETH Zurich Email author 
  • , Markus BeckAffiliated withNanotechnology Group, Department of Mechanical and Process Engineering, ETH Zurich
  • , Reto FiolkaAffiliated withNanotechnology Group, Department of Mechanical and Process Engineering, ETH Zurich

Abstract

Widefield fluorescence microscopy is seeing dramatic improvements in resolution, reaching today 100 nm in all three dimensions. This gain in resolution is achieved by dispensing with uniform Köhler illumination. Instead, non-uniform excitation light patterns with sinusoidal intensity variations in one, two, or three dimensions are applied combined with powerful image reconstruction techniques. Taking advantage of non-linear fluorophore response to the excitation field, the resolution can be further improved down to several 10 nm. In this review article, we describe the image formation in the microscope and computational reconstruction of the high-resolution dataset when exciting the specimen with a harmonic light pattern conveniently generated by interfering laser beams forming standing waves. We will also discuss extensions to total internal reflection microscopy, non-linear microscopy, and three-dimensional imaging.

Keywords

Structured illumination Extended resolution Widefield fluorescence microscopy