Super-resolved Microscopy

Abbe’s statement of the diffraction-limited resolution achievable in a microscope given by ~λ_em/(2 NA) was widely perceived since 1837 as a fundamental limit. To image smaller features than could be discerned in optical microscopes, it was believed to be necessary to use much shorter wavelength radiation in, e.g. X-ray microscopes or electron microscopes (noting the de Broglie wavelength of an electron is given by λ = h/mv). Such radiation is typically damaging for most biological samples and usually precludes live imaging. It has recently been shown, however, that the diffraction limit can be circumvented such that biological samples can be imaged with sub-50 nm resolution using visible optical radiation in the far field. Super-resolved microscopy (SRM) is now a very dynamic field and was recognised by the Nobel prize for Chemistry in 2014 to Stefan Hell, W. E. Moerner and Eric Betzig. This recognition is appropriate because the super resolution was achieved by manipulating the emission from fluorophore labels – essentially switching it on and off, either in a spatially structured manner on a scale below the diffraction limit or stochastically such that the emission from individual fluorophores can be localised.

The first established SRM approach was stimulated emission depletion (STED) microscopy^{2, 3}, which is a super-resolved analogue of laser scanning confocal microscopy and involves using the nonlinear suppression of fluorescence through depletion of the excited state population to confine fluorescence emission to a region significantly smaller than the PSF. This approach can be extended to any controlled switching of fluorescence and the general approach has been described by Stefan Hell, its pioneer, as “reversible saturable optical (fluorescence) transitions” (RESOLFT)³. Other RESOLFT systems include the use of reversibly switchable fluorophores including fluorescent proteins^{4, 5} which have provided sub-40 nm resolution. We have worked extensively on STED microscopy, developing microscopes that incorporate programmable compensation of optical aberrations and combine super resolution with FLIM. Most recently we have incorporated these concepts in a 3-D STED microscope.

The second main class of SRM techniques, for which W.E. Moerner and Eric Betzig were honoured, is based on the stochastic switching of the emission of individual fluorophores such that they can each be localised⁶ to a precision beyond the diffraction limit. The localisation of individual fluorophores is readily implemented in TIRF microscopes and the stochastic switching can be implemented optically using photoswitchable fluorophores, for which most common techniques for such localisation microscopy are described as photoactivated localization microscopy (PALM)^{7, 8} when using photoswitchable fluorescent proteins and stochastic optical reconstruction microscopy (STORM)⁹ when using pairs of dye fluorophores. STORM localisation microscopy has been extended to chemically-induced switching of single fluorophores under continuous excitation and is described as d-STORM¹⁰. PALM, STORM and related techniques have both provided sub-10 nm lateral resolution of biological samples and have been extended from fixed cells to live cell imaging^{11, 12}. We are working on making localisation microscopy simpler and have recently demonstrated easySTORM¹³, which can be implemented on standard TIRF or wide-field fluorescence microscopes using low-cost components and open source software.