Imaging has long been recognized as a crucial tool for understanding cellular structure and function. Diffraction has however limited light microscopy to a resolution of 200nm at best, leaving the observation of ultrastructural cellular features to the field of electron microscopy (EM). This includes cellular structures that contribute to cell motility, force generation and mechanosensing such as actin filaments, microtubules and focal adhesion complexes.

Advances in optical techniques, broadly termed super-resolution fluorescence microscopy, together with advances in fluorescent labeling methods, have extended the resolving power of light microscopy towards the nano-scale, as is described in recent reviews  [1, 2, 3, 4]. These advances make it possible to dissect the molecular architecture of cells without subjecting them to EM processing techniques.

Diffraction is a manifestation of the wave properties of light. In a typical light microscope operating in the visible spectral range (400-700 nm) (see Figure below), diffraction determines the smallest focal volume that light can be focused into. This is referred to as the point spread function (PSF) and limits resolution to ~250 nm in the X-Y image plane and ~500 nm along the Z optical axis [2]. In other words, in conventional light microscopes such as confocal or TIRF (Total Internal Reflection Fluorescence), structural features lying closer to each other than the PSF length scale cannot be resolved. This limitation has however been over come with the development of various super resolution microscopy methods.