Super-resolution Methods

The resolution of conventional light microscopes (wide-field/confocal microscopes) is limited by the diffraction of light.

On a practical level, this is generally understood to set a theoretical limit for the horizontal resolution of a light microscope with a good objective to approximately half the wavelength of light used (so ca. 250 nm in green, 350 nm in deep red).

According to this interpretation of super-resolution the methods available at BIU are:

Structured Illumination
- Enhances spatial resolution by superimposing a pattern of illumination on the imaged area and collecting a sequence of images while shifting the pattern. Knowledge of the pattern and shift is then used to collect high-frequency information from outside the observable region.
- XY resolution is approximately double that of a conventional light microscope (120 nm in green)
- Instruments at the BIU:
  - GE DeltaVision OMX SR
Localization microscopy
- Images reconstructed by sequentially activating sparse subsets of fluorophores and modeling the centroid position of each fluorophore activated. Final images are constructed by combining the localization of many (thousands) of images.
- STORM, dSTORM, PALM,...
- XY resolution of the final image is dependent on the localization accuracy and mathematical model used but can be better than 50 nm
- Instruments at the BIU
  - GE DeltaVision OMX SR
- https://www.microscopyu.com/techniques/super-resolution/single-molecule-super-resolution-imaging
- https://www.nikoninstruments.com/en_EU/Products/Super-resolution/N-STORM-Super-Resolution
AiryScan
- XY resolution 140 nm
- Z resolution 400 nm
- Zeiss LSM 880
- http://www.nature.com/nmeth/journal/v12/n12/full/nmeth.f.388.html
HyVolution & Lightning
- Confocal methods combining pinhole adjustment with advanced deconvolution
- XY resolution 140 nm
- Leica TCS SP8X
- Leica Stellaris 8
Total Internal Reflection (TIRF)
- Fluorescence excitation by an evanescent wave at the cover slip. Super-resolution is in the axial direction directly at the coverslip surface (For example the plasma membrane of adherent cells).
- <200 nm thick imaging region
- GE DeltaVision OMX SR (Ring-TIRF)
- https://en.wikipedia.org/wiki/Total_internal_reflection_fluorescence_microscope