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Click on the images below to compare the resolutions possible using different super-resolution techniques. Access charge is £30.42/hr.

Single Molecule Localisation Microscopy (SMLM)

SMLM techniques like Photoactivation Localisation Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) rely on sparse visualisation of a small subset of fluorescent emitters. This is achieved in different ways depending on the technique. For example, in PALM, photo-activatable or photo-switchable fluorophores are used and only a few of them are converted to the emitting state at any one time, whereas in STORM the fluorophores are driven into a dark state and stochastically return to an emitting state. In both cases the images are made up of spots that blink on a frame-by-frame basis. Each spot represents an individual emitter. Capturing many thousands of these images and localising the centre of each spot allows one to reconstruct an image made up of each localised molecule. The precision of localisation depends upon the specimen and how well the experiment has been optimised, but under ideal conditions it can be less than 10 nm.

Once molecules have been localised their clustering can be analysed using such programs as SR-Tesseler. For SMLM at the LMCB, use the Carl Zeiss Elyra PS.1. In addition the ONI Nanoimager can be used for PALM/STORM and also single molecule tracking and smFRET.

Instant SIM (iSIM)

Instant SIM is a technique pioneered by York et al and developed commercially by Visitech International Ltd. The concept uses the Image Scanning Microscopy (ISM) approach described mathematically by Colin Sheppard in 1988. The technology creates a super-resolution image in analogue form using optics alone so that the image produced by the camera requires no reconstruction, although deconvolution improves the resolution further.

For iSIM at the LMCB, use GFP4.

Airyscan

Airyscan is another ISM technique developed by Carl Zeiss and implemented on the company's LSM confocal microscopes.

iSIM, Airyscan and other image scanning microscopy techniques approach the theoretical maximum resolution possible using confocal microscopy but do not break Abbe's diffraction limit, which limits the resolution of light microscopy to approximately half the wavelength of light. For Airyscan at the LMCB, use the LSM 900 inverted or LSM 900 upright.

Structured Illumination Microscopy (SIM)

In Structured Illumination Microscopy high frequency information that is out of the bandwidth of the diffraction-limited microscope optics is shifted into the bandwidth by convolution with a known frequency; normally a grid pattern projected using a diffraction grating or spatial light modulator. The grating pattern must be shifted laterally and rotated in order to sample high frequencies in all orientations, which means 15 to 25 images must be taken to make one super-resolution image.

Its ability to access higher frequencies means SIM breaks the diffraction limit and doubles the resolution of light microscopy. However, SIM is slower than either iSIM or Airyscan and it is not confocal, so is not ideal for thick specimens with lots of out of focus light. For SIM at the LMCB, use the Carl Zeiss Elyra PS.1.

Stimulated Emission Depletion (STED)

In Stimulated Emission Depletion a (usually) doughnut-shaped depletion laser is used to stimulate re-emission of excitation photons from a region around the periphery of the excitation spot of a laser scanning microscope, effectively making the excitation spot smaller and allowing the microscope to scan at a higher resolution. Resolution increases with depletion laser power and very high laser powers are needed. Time-gating of the detectors improves resolution further by eliminating the small amount of fluorescence emission in the region of the doughnut, which makes it possible to reduce the depletion laser power.

For STED at the LMCB, use the Leica STED.