Sub-100 nm 3D Detection Volumes by Total Internal Reflection STED Microscopy

Abstract

Stimulated Emission Depletion (STED) Microscopy (1–2) has revolutionized far-field fluorescence microscopy by breaking the classical diffraction limit: 25 nm resolution and better are routinely achieved in the focal plane.Fig. 1. 20 nm fluorescent bead imaged in confocal and STED mode. The profile across the white box demonstrates <26 nm resolution.View Large Image | View Hi-Res Image | Download PowerPoint SlideWhile comparable axial resolution values have been obtained using two opposing objectives, single-objective STED microscopy can usually not realize values on this size scale.Here we present results obtained with a recently realized STED variant that combines total internal reflection excitation for ∼70 nm axial sectioning capabilities with stimulated depletion for ∼50 nm lateral super-resolution.TIRF STED microscopy represents an attractive super-resolution alternative for live cell microscopy featuring fast scanning with sub-100 nm 3D detection volumes and reduced photo-damage through TIRF excitation.