STED microscopy

Stimulated Emission Depletion microscopy, or STED microscopy, is a technique that has overcome the limits imposed by diffraction with standard confocal laser scanning microscopes and conventional far-field optical microscopes ^[1].

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A confocal laser scanning microscope uses a focused laser beam to illuminate a small part of the sample being observed. The laser is tuned to a frequency that excites fluorescence from a dye in the sample, and light from the small region being excited is observed by a detector. The resolution of a confocal scanning microscope is limited to the spot size to which the excitation spot can be focused. This size depends on system parameters, but is typically approximately the wavelength of the excitation beam.

Stimulated Emission Depletion microscopy reduces the size of the excited region by using a very short excitation pulse which is immediately followed by a "depletion" pulse, tuned to an emission line of the fluorescent dye. This depletion pulse causes stimulated emission, moving electrons from the excited state (from which fluorescence occurs) to a lower energy state. The wavefront of the depletion beam is altered in such a way that it is focused to a ring instead of a spot, featuring a dark spot of zero laser intensity in the center. While this dark spot is itself diffraction-limited, the intensity distribution is continuous and is zero only at the center. Therefore, using a bright depletion pulse causes almost all of the electrons excited by the excitation pulse to return to the ground state, leaving only the region of the sample very close to the axis of the depletion beam excited. After both pulses have been sent, fluorescence from the remaining excited dye molecules is detected by the microscope. As of 2006, resolution improvements over confocal laser scanning microscopy of up to 12-fold have been reported^[2].

STED is the first implementation of a more general concept known as RESOLFT and under ideal conditions, the improvement of the spatial resolution is given by an equation common to all methods based on the concept. Another RESOLFT-based technique proposed early on was Ground State Depletion microscopy^[3], which uses an excitation pulse to boost off-axis ground-state electrons to a long-lived higher energy state before exciting remaining ground-state electrons with a fluorescence excitation pulse.

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