Stimulated Emission Depletion Microscope (STED)

Since the introduction of confocal microscopy in the ‘80s, advanced light microscopy became indispensable in life science research. The wish to ever see more details has driven new developments in light microscopy. Although, the laws of physics predicted a fundamental limit to the maximal achievable optical resolution, Dr. Stephan Hell was one of the first to realize that this limit might not be as unsurpassable as it was believed to be. Thanks to advances in fluorescent labels and laser technology, he and others demonstrated that it was possible to visualize beyond the resolution limit of light with a diverse set of technologies that are now referred to as ‘super resolution microscopy’ or ‘nanoscopy’.

Perhaps the most applicable form of 'nanoscopy' is Stimulated Emission Depletion (STED) nanoscopy which makes use of two coinciding laser beams that are scanned across the sample to generate an image. The first laser beam serves, as with confocal microscopy, to excite the fluorophores, while the second laser beam has a ‘donut’ shape which suppresses fluorescence except in the centre. This generates an effective resolution of 30 - 80 nm, similar to the size of e.g. a ribosome.

STED nanoscopy is ideal for samples were structures cannot be separated because of the resolution limit. All samples compatible with confocal microscopy can be used provided that the labels, coverslip (#.15) and refractive index of the sample are compatible with STED.

The microscope is equipped with three objective lenses, one for confocal imaging (20X, NA 0.8) and two for STED imaging with different refractive indexes (60X, oil, NA 1.42 and 60X silicone oil, NA 1.3). STED can be performed with dyes compatible with the 775 nm or 595 nm pulsed depletion laser and the four excitation lasers (405 nm (CW), 485 nm (pulsed), 561 nm (pulsed), 640 nm (pulsed)). Detection is performed by single photon counting APD point detectors or a Matrix detector allowing to reduce background signals.

A resolution improvement is possible in both axial as lateral direction using the 3D beam shaping module. Fluorescence lifetime observation is possible. A Z-drift compensation system and transmission and epi-fluorescence illumination systems are present.

Live samples are possible using the cage incubator for CO2, temperature and humidity control. Light exposure can be minimized by an adaptive illumination scheme to reduce photobleaching and toxicity.