The Wolfson Bioimaging Facility offers a few ways to achieve fluorescence imaging below the 200nm resolution limit of conventional light microscopes. Although broadly termed ‘super-resolution’, these approaches vary in the resolution achieved and some may more accurately be described as providing ‘enhanced’ or ‘optimised’ resolution. More thorough comparison of the available techniques is provided on additional pages.
Deconvolution of optimally acquired image stacks enables some improvement in resolution and when combined with closing of the confocal pinhole on CLSM systems or additional optics on a spinning disk system can reach the theoretical resolution limit of confocal microscopes (~120-140nm).
Leica’s ‘HyVolution’ approach integrates Huygens deconvolution software on one of our confocal microscopes. This global deconvolution approach has been superseded by Leica’s ‘Lightning’ adaptive deconvolution process available on our three most recently acquired CLSM systems.
The Yokogawa CSU-W1 SoRa super-resolution spinning disk provided as part of our Olympus IXplore SpinSR system similarly approaches the resolution limit of an ‘ideal’ confocal microscope (~140nm) and with additional deconvolution can achieve ~120nm resolution. The higher sensitivity and speed of this system is more amenable to live imaging with enhanced resolution than CLSM approaches.
STED (Stimulated emission depletion) is a true optical super-resolution technique in which a doughnut shaped laser beam silences fluorescence around a central excitation spot and thereby diminishes the excitation volume of a CLSM system to below the conventional diffraction limit. Our gated 3D STED system enables super-resolution imaging of some green and red fluorophores to ~50 nm xy and ~150 nm z resolution.
Single molecule localisation nanoscopy methods such as STORM, PALM and PAINT push resolution to <20nm by localising single fluorescence events. These techniques are facilitated together with conventional TIRF imaging by our Abbelight SAFe360 system on Olympus IX83 microscope.
Specifications of our enhanced and super-resolution systems are provided in the instrument data documents:
- Leica SP8 3D STED (also with HyVolution)
- Leica SP8 Confocal 12 with Lightning
- Leica SP8 Confocal 13 with Lightning
- Leica SP8 Confocal LSB-2 with Lightning
- Olympus SpinSR – super-resolution spinning disk
- Olympus/Abbelight SAFe360 Single molecule/TIRF system
Recent publications with super-resolution data include:
Hellewell, Heesom, Jepson, Adams (2022) PDIA3/ERp57 promotes a matrix-rich secretome that stimulates fibroblast adhesion through CCN2 American Journal of Physiology – Cell Physiology doi: 10.1152/ajpcell.00258.2021 DOI: 10.1002/nano.202100183
Samphire, Takebayashi, Hill, Hill, Heesom, Lewis, Alibhai, Bragginton, Dorh, Spencer, Galan (2021) Green fluorescent carbon dots as tartgeting probes for LED-dependent bacterial killing. Nano Select DOI: 10.1002/nano.202100183
Tian, Zhang, Du, He, Jin, Pearce, Eloi, Harniman, Alibhai, Ye, Phillips, Manner (2020) Tailored self-assembled photocatalytic nanofibers for vsisible-light-driven hydrogen production. Nature Chemistry 12(12):1150-1156
Tan, Alibhai, Cross, Thompson, Wulfing (2020) Super-resolution imaging of the T cell central supramolecular signaling cluster using stimulated emission depletion microscopy. Bio. Protocol. 10(21):e3806