Abberior Facility STED Super-resolution/Confocal Microscope

The development and subsequent availability of various super-resolution fluorescence microscopy techniques in the past decade has enabled researchers to perform sub-diffraction-limit (i.e., to overcome the 200 nm diffraction limit) imaging of ultrafine biological structures. The Nikon A1/SIM/STORM super-resolution/confocal microscope managed by the ULS allows users to perform structured illumination microscopy (SIM) and stochastic optical reconstruction microscopy (STORM). However, SIM only improves the resolution by a factor of two (to ~100-115 nm), and is known to introduce imaging artefacts in certain samples. STORM, on the other hand, is not compatible with living samples. In addition, both techniques are not suitable for imaging biological tissues or other thick specimens. To address the pressing need to adopt super-resolution microscopy for live-cell and animal-tissue imaging, the ULS has acquired a new generation of stimulated emission depletion (STED) super-resolution microscope with the support from the RGC Collaborative Research Fund (2020/21).

The principle of STED is rather simple and truly optical. Fluorophores at the outer region of the excitation beam are “switched off” by a doughnut-shaped depletion beam. Thus, only signals at the centre of the excitation beam (which are sub-diffraction limit) are detected. The Abberior Facility STED super-resolution/confocal microscope further enhanced the STED technique by a few approaches, including time-gated signal detection to improve lateral resolution (i.e., gated STED), a second depletion light path to improve axial resolution, adaptive illumination to minimise photodamage, as well as adaptive optics (AO) to reduce aberrations in relatively thick samples. These new features fill an important gap between SIM and STORM, and would allow researchers to perform live-cell as well as animal-tissue super-resolution imaging with best lateral and axial resolution of 30 nm and 70 nm, respectively.