The resolution barrier imposed by the diffraction of light has long been a major obstacle in light microscopy. Diffraction limits the behaviour of light in optical microscopy, setting the shortest achievable distance to approximately half the wavelength of light (>200 nm). Here, resolution is defined as the shortest distance between two points before they blur and become indistinguishable from each other. With a diverse range of fundamental cellular processes occurring in the sub-wavelength range at the macromolecular level (i.e. from a few nanometers to a few hundred), they have been beyond the reach of traditional light microscopy.
Super resolution microscopy aims to break through this 200 nm barrier and in doing so can reveal cellular processes and dynamics previously unseen. By combining super resolution techniques with fluorescence we are able to pair this improved resolution with highly specific, multi-colour and live cell imaging.
New methods of achieving improved resolution have been developed over the past decade based on different underlying principles. STORM and PALM are a type of single molecule localisation microscopy, whereas iSIM is a type of structured illumination microscopy that relies on analog improvements to the optical system itself rather than digital image processing operations.
These techniques rely on localising single fluorescent molecules with high precision by determining the centre of their point-spread functions (PSF). The PSF is the 3D diffraction pattern of light emitted by an individual fluorescent molecule.
STORM employs the photoswitchable, aka ‘blinking’, properties exhibited by some fluorophores. Allowing a different subset to be ‘switched on’ at any one time allows for temporal separation of the fluorescent probes that would otherwise spatially overlap and render localisation of their respective centres difficult. Many imaging cycles are conducted, in which fluorophores are activated, imaged and deactivated, with each cycle capturing a different sub-population of the fluorescent probes. The centres of all of these fluorescent ‘blinks’ can be determined and then reconstructed to form a super resolution image.
PALM relies on a very similar premise except that it was developed utilising endogenously expressed fluorophores, such as fusion constructs with a photoactivatable fluorescent protein tag (whilst STORM was developed using antibodies conjugated to an organic fluorophore).
The iSIM is a novel structured illumination microscope that allows rapid (up to ~100 frames per second) imaging at double the normal resolution of a wide field microscope (e.g. ~150 nm resolution for GFP tagged proteins), with high contrast.
It works on the basis of illuminating the sample with an array of micro lenses (the ‘structured illumination), and recovering the emitted light through pinholes that are shifted by a distance of X. This reduces the width of the point spread function, allowing the improvement in resolution. As this image is shifted by X/2, a second array of micro lenses then shifts the signal back to its true position, and all the signals are summed from the back shifted pinhole positions and summed to generate the super-resolution image. The final image (already super-resolved) is collected by a CMOS camera. Deconvolution can then be used to improve resolution to a full 2-fold improvement.
The technique can be used for optical sectioning in relatively thick specimens, either in-vivo in small animal models or in-vitro with excised tissue or fixed tissue sections. Depths of up to 1 mm can be achieved and with the addition of clearing agents up to 2 mm.
FLIM (fluorescence-lifetime imaging microscopy)
FLIM produces an image based on the differences in the excited state decay rate of a fluorescent sample. Two-photon excitation combined with Time-Correlated Single Photon Counting (TCSPC) are used to determine the fluorescence lifetime in each pixel. Applications of this technique include the study of molecular interactions by FRET and changes in a variety of environmental parameters.
With down to 140 nm lateral resolution, the LSM880 with Airyscan can be included as a type of super resolution microscope.
This improvement in resolution is achieved by the use of a multichannel area detector with 32 elements –each detector element functions as a single pinhole and allows more light to be collected. This differs from a classical confocal microscope, which illuminates one spot on the sample and employs a single pinhole to reject out of focus light.
The size of the pinhole determines how much light reaches the detector, so whilst a smaller pinhole increases resolution, it also means less light gets through and the signal-to-noise ratio (SNR) decreases significantly. By using a series of pinholes, more light is collected in total, maintaining a good SNR, whilst the resolution can be improved by using 32 smaller pinholes in the detector.
For a more in depth description of how the Airyscan works, click here, to visit the Zeiss page.