Super-resolution light microscopy
Part of the Wolfson Light Microscopy Facility
Super-resolution microscopy is a form of fluorescence light microscopy, which allows the capture of images with a resolution below that of the Abbe diffraction limit. There are numerous different ways to achieve this leading to a broad variety of methods such as 4Pi microscopy, structured illumination microscopy, stimulated emission depletion (STED), reversible saturable optical fluorescence transitions (RESOFLT), STORM, PALM, to name a few. Achieving a spatial resolution that is not limited by the diffraction of light allows for the observation of many biological structures not resolvable in conventional fluorescence microscopy. The Wolfson Microscopy Facility has three instruments allowing imaging with a resolution below the diffraction limit.
SIM – Structured Illumination Microscopy
Structured Illumination Microscopy (SIM) is based on the use of patterned illumination. Using structured (patterned) illumination, rather than standard uniform illumination, generates an image in which information from the sample and the pattern are combined, bringing small variations in sample fluorescence within the diffraction limit of the microscope. By using multiple different patterns on the same area of sample and with precise knowledge of what those patterns are, software can be used to reconstruct a super-resolution image. Possible resolution: two-fold improvement compared to diffraction limited imaging (wavelength dependent) ~ 120nm (xy) and ~320nm (z). The system is ideal for cultured cells and brightly stained samples and can be used for live cell imaging under certain conditions.
System specification OMX:
DeltaVision/ GE OMX optical microscope (version 4) for structured illumination (3D-SIM), UltimateFocus Hardware Autofocus Module, x,y,z, nanomotion sample stage with fast piezo -z-axis sytem; 6 colour widefield illumination module and Ring TIRF; temperature and CO2 controlled; software: DeltaVision OMX softWoRx 6.0
Laser lines: 405nm, 488nm, 568nm, 642nm;
Lenses: 60x 1.42 oil planapochromat; 60x Apo TIRF 1.49 oil
- 436/31 (e.g. DAPI)
- 528/48 (e.g. Alexa Fluor 488)
- 609/37 (e.g. Alexa Fluor 568, mCherry)
- 683/40 (e.g. Alexa Flour 647, Cy5)
Further information on the OMX and SIM can be found in the downloads above (authors: Ian Dobbie and Lothar Schermelleh, Oxford). More information can be found on our intranet page.
The system can also be used for FRAP experiments and for TIRF imaging (ring TIRF set up).
Projects run on the OMX so far include:
- novel stains for organelles
- cell division in bacteria
- Weibel Palade bodies formation and secretion
- difference in bacterial cell volume in a number of knock out strains
STORM: Stochastic Optical Reconstruction Microscopy/ PALM: Photo-Activated Localisation Microscopy
PALM/STORM imaging relies on specific characteristics of the fluorophores used. Initially, all fluorophores are (effectively) in an off state. When imaging, a random subset of fluorophores are switched on and imaged before being driven back to an 'off' state. As only subsets of fluorophores are imaged at the same time and these are far apart and easily resolvable from each other, their locations can be approximated and recorded. This is carried out for many (>1000) frames allowing a high-resolution image to be formed from all single molecule fluorophore positions calculated. N-STORM uses dual fluorophore- labelled antibodies (labelled with activator (e.g. AF405, Cy3) and reporter (eg AF 647, Cy5)), dSTORM utilises reporter only labelled antibodies. PALM relies on photo-switchable/ - activatable fluorescent proteins. Possible resolution: ~30nm (xy) and ~ 50nm (z), ideal for fixed cells/ molecular structures.
System specification N-STORM:
Ti-NS N-STORM with 3D capability, motorised stage controller with piezo insert, petri dish holder and slide holder for stage, temperature controlled incubator, EM-CCD camera, separate PC for image reconstruction work;
Laser lines: 405nm, 488nm, 561nm, 647nm;
Lenses: SR Apo TIRF 100x; CFI Plan Apo TIRF 60x oil
Filterset: Quad cube for blue/green/red and far red; eYFP and red (optimized for mEOS)/ far red combination also available
Please see also the information in the download above and on our intranet page.
Projects run on the N-STORM so far include:
- cell division in bacteria
- tetrahymena thermophile as a model organism for cilia
- localisation and clustering of tetraspanins
- cellular polarity in embryo development
- correlative approach combining STORM with Atomic Force microscopy (AFM)
- Lakadamyali (2014) ChemPhysChem, 15:630-636;
- Dempsey et al. (2012) Nature Methods, 8:1027-1036
- Lippincott-Schwartz & Patterson (2009) Trends in Cell Biology, 19:555-565
- Schermelleh et al. (2010) J Cell Biol, 190:165-175
The AiryScan is a confocal scanning microscope with an additional detector. This allows the AiryScan to detect fluorescent signals with a concentrically-arranged hexagonal detector array. Each array acts as a small pinhole which allows the confocal pinhole to remain open allowing the collection of the extended Airy disk from each fluorescent point in the sample. The signals from the array are then re-assigned to their correct position/ the original fluorescent point. This yields an image with an improved signal to noise ration and increased resolution. Achievable resolution is in the range of 120nm in xy and 350nm in z. In addition with the fast mode it is possible to acquire 2 fps (480 x 480 pixels). In confocal mode 5 fps (512 x 512 pixels) are possible.
System specification Zeiss AiryScan
Zeiss LSM 880 invert based on Axio Imager.Z with an environmental control chamber, FCS capability, fast piezo Z, defined focus system, GaAsP detector and galvo scanner
Laser lines: 405nm, 458nm/488nm/514nm argon laser, 561nm, 633nm
Lenses: 5x, 10x, 20x, 40x water, 40x oil, 63x oil
Filterset: full set of filter for AiryScan detector
Detectors: AiryScan, GaAsP, two PMT, spectral detector in confocal mode allowing to tune the emission collection
There is also more information on the AiryScan on our intranet page.
- General AiryScan principle
- AiryScan instrument description
- Mueller and Enderlein (2010) Physical Review Letters 104, 198101, DOI: 10.1103/PhysRevLett.104.198101