STED nanoscopy

Abstract

This chapter provides an introduction to the fundamentals of STED nanoscopy. The first section starts with a discussion of the field and intensity distribution in the vicinity of the geometric focus by means of vectorial diffraction theory. In a next step, we introduce the fluorescence microscope, deduce the formulas for the classical resolution limit for incoherent image formation and discuss the implications for confocal fluorescence microscopy. In the second section, the principle underlying STED nanoscopy is introduced and the fundamentals necessary for its further understanding are discussed. First, we cover the basic photophysical interactions between fluorophores and the excitation and STED light. On this basis, we derive boundary conditions for the light sources to be used. We then discuss the dependence of fluorescence suppression on the relevant photophysical and experimental parameters. Finally, we explain the mode of operation of the phase patterns used for shaping the STED beam and discuss the achievable resolution. Lastly, we show in the third section examples of applying STED microscopy to cell imaging.