STED microscopy, or stimulated emission depletion microscopy, is a superresolution imaging technique in fluorescence microscopy that surpasses the diffraction limit, enabling the visualization of structures at the nanoscale level. This technique was developed to overcome the limitations imposed by the diffraction of light, which traditionally hindered the resolution of optical microscopy to a few hundred nanometers.
Key features and principles of STED microscopy:
Superresolution: STED microscopy achieves superresolution by using a pair of laser beams — a stimulating beam and a depleting beam. The stimulating beam excites fluorophores in the sample, while the depleting beam suppresses fluorescence from the outer regions, confining the excitation to a smaller region than is achievable with conventional microscopy.
Stimulated emission: The depleting beam is tuned to a wavelength where it can stimulate the emission of fluorescence from the excited state of the fluorophores. By spatially and temporally overlapping the stimulating and depleting beams, the effective excitation region is reduced to a much smaller volume than the diffraction limit.
Fluorescent molecules: STED microscopy typically uses fluorescent molecules that can be switched between a dark state and a bright state. This switching, combined with the spatially controlled depletion of fluorescence, allows for the precise localization of individual fluorophores.
Point spread function: The point spread function in STED microscopy is engineered to be much smaller than the diffraction limit, allowing for the resolution of features as small as a few tens of nanometers.
Applications: STED microscopy has been widely used in biological research to study cellular structures and processes at a level of detail previously unattainable with conventional microscopy. It is applicable to various samples, including live cells, tissues, and other biological specimens.
Multicolor imaging: STED microscopy can be adapted for multicolor imaging by using different fluorophores that respond to specific wavelengths of light. This allows researchers to visualize multiple cellular structures or molecules simultaneously.
Advancements: Continuous advancements in STED microscopy technology have led to improved spatial resolution, reduced photobleaching effects, and increased imaging speed, making it a powerful tool in the field of superresolution microscopy.