The Principle, Application and Imaging of STED

Abstract

In a conventional optical microscopy system, due to the diffraction effect of the optical element, the spot light formed on the sample after the parallel incident illumination light is focused by the microscope objective is not an ideal point, but a diffraction spot having a certain size. Samples within the diffraction spot will fluoresce as a result of illumination by the illumination, such that sample detail information within the range is not resolved, thereby limiting the resolution of the microscopy system. In order to overcome the limitations of the diffraction limit and achieve super-resolution microscopy, how to reduce the effective fluorescent light-emitting area at a single scanning point becomes a key. In STED microscopy, the reduction in effective fluorescent luminescence area is achieved by stimulated emission effects. In a typical STED microscopy system, two illuminations are required, one for excitation and the other for loss of light. When the excitation light is irradiated so that the fluorescent molecules in the diffraction spot range are excited, and the electrons transition to the excited state, the loss light causes some electrons outside the excitation spot to return to the ground state in a stimulated emission manner, and the rest is located in the excitation spot. The excited electrons in the center are not affected by the loss of light and continue to return to the ground state by autofluorescence.