Superresolution Fluorescence Microscopy
Superresolution Fluorescence Microscopy is broadly used to describe techniques that surpass the optical diffraction limit in one or more dimensions (Dr ~ l/2.NA in the lateral direction, and Dz ~ 4 Dr/NA in the axial direction; where NA = Numerical Aperture, l = wavelength). Usually they employ using some extra degree of freedom, such as selective illumination of sample with a pattern (structured illumination) or to partially deplete excited fluorophores and reduce emission volume (stimulated emission), or selectively or stochastically excite discrete subsets of fluorophores and based on the optical performance of the system in response to a point emitter determine a more accurate position of the discretely emitting molecules (“localization microscopy”). Alternatively, to achieve improved axial resolution certain physical principles such as attenuated total internal reflection (TIR) , interference effects or spectral encoding may be employed. Depending on the technique special sample preparations and fluorescent labelling may be required, and imaging is in all cases not as fast as “non-”superresolution imaging and hence more challenging on live cells.