Introduction

STED (Stimulated Emission Depletion) microscopy is one of the many different types of super-resolution microscopy techniques which have been recently developed to increase spatial resolution through exceeding the limit of diffraction of conventional light microscopy. STED microscopy allows to obtain super-resolution images by the selective deactivating fluorophores, minimizing the area of illumination at the focal point, and enhancing the achievable resolution for a given system. It finds applications in the study of sub-cellular architectures and dynamics and where it takes advantage of the non-linear response of fluorophores commonly used for marking biological samples. Thou STED microscopy is widely used and successful, there is still room for improving the optical resolution of the acquired images.

Technical features

Presented is a method for increasing the optical resolution of a STED microscope based on the modulation of the intensity of its beam on an arbitrary time scale during image acquisition, without increasing the intensity of the STED beam and in a simple and economic manner. Such method is based on the principle that the fluorescence at the centre of an observation volume is not modulated, while the fluorescence at the periphery of said volume is modulated. The exploitation of this difference in the modulation of the fluorescence signal entails an increase in the spatial resolution.

Possible Applications

• Image analysis of cells, living tissue and organisms;
• Structural analysis of non-biological materials.

Advantages

• Increased resolution of STED microscope;
• Does not require to increase the intensity of the STED beam;
• Reliable, economical and easy to implement.