Nikon N-SIM / N-STORM Super Resolution Microscopy (Baddiley-Clark Building)
Background: The resolution of any optical system is limited by its component parts and the wave-like nature of light. Furthermore, the sample itself may provide a resolution- limiting barrier that must be overcome; even with the best available microscope this may not be possible. This is a problem for biologists who need to probe structures, individual proteins, and protein complexes beyond the limits of currently available ‘conventional’ optical systems.
Going smaller brings us into the domain of electron microscopy (EM) but not all biological systems are amenable to EM analysis. Super resolution microscopy extends the resolving power of the light microscope into that previously achieved using EM. SIM (Structured Illumination microscopy) and Pointillism (STORM (STochastic Optical Reconstruction Microscopy) are two super resolution microscopy techniques that are capable of transending the diffraction limited resolution barrier of conventional microscopy.
N-SIM
Structured illumination microscopy systems such as the Zeiss ApoTome project a grid (positioned in the field diaphragm plane of the fluorescence illumination axis) pattern over the specimen. This grid pattern is translated over the specimen by tilting it back and forth in the light path. At least three images of the specimen are acquired with the grid in different orientations. The grid pattern and with it the out of focus light is computationaly removed from the final image. The result is similar to laser scanning confocal microscopy in terms of resolution and optical section thickness.
In a similar way to the Apotome approach the SIM technique involves projecting a series of ‘high frequency’ striped patterns (using an optical grating) onto the specimen. Moiré fringes containing information relating to the specimen’s sub resolution structure develop when this pattern illuminates finer labeled structures of the sample. This information is extracted by image processing algorithms and a super resolution image is formed by combining multiple images collected from different grating orientations. With SIM, one can expect to roughly increase the resolving power by a factor of two (~100 nm). Importantly, because the technique is not reliant on the properties of the fluorescent probe nor requires special sample preparation, it is possible to image most fluorescent labels under typical IF conditions.
N-STORM
Seurat and exponents of the pointillism paining technique, used individual dots to form a picture; a similar approach is used by single molecule imaging techniques such as STORM to visualize biological structures beyond the resolution of conventional light micrsocopy. Such techniques exploit the properties of the fluorophore itself in particular, its ability to be photo-activated, bleached or photo-switched.
Conventional STORM imaging (N-STORM) uses photo-switchable fluorophore-labeled antibodies (antibodies are labelled with activator (eg AF405, Cy3) and reporter (eg AF647, Cy5) fluorophores) to densely label structures or proteins of interest. Initial irradiation with high-intensity light ensures that all fluorophores are converted to an ‘off’ or dark non-fluorescent state before image capturing commences. When imaging, a random subset of fluorophores are switched on and imaged before being driven back to their ‘off’ state. As long as the distance between each fluorophore is larger than the resolution of the system, the center of their point spread function (PSF) can be approximated and recorded. On and off cycles are repeated many (>1000) times allowing a high-resolution image to be formed from all single molecule fluorophore positions calculated. A variant of STORM known as direct or d-STORM utilises reporter only labelled antibodies.
Imaging modes: Super resolution (N-SIM & N-STORM)
Resolution and acquisition rate: N-SIM: lateral resolution 85-110 nm (optics and imaged wavelength dependent), Axial (z) resolution: 200-250 nm (optics and imaged wavelength dependednt), Axial range (3D-SIM ) 20 micro m. Image acquisition rate: 2D SIM 1.6 fps, 3D SIM 1 fps. N-STORM: lateral resolution 20-30 nm, axial (z) resolution 50-60 nm, 3d STORM 50-75 nm. Image acquisition rate: typically 5-30 minutes.
Laser lines: N-SIM: 457, 488, 515, 532, 561. N-STORM: 405, 457 488 514, 561, 647
Objectives: 100x Apo TIRF (oil, 1.49)
Location
Centre for Bacterial Cell Biology (Baddiley Clark Building) room 4.25
Local contact: Dr Richard Daniel ext 3239
Booking and Availability
Please contact Alex Laude 246 4860
