Life sciences require the third dimension with high spatial and temporal resolution

A recognized advantage of optical microscopy lies in the fact that allows non-invasive three-dimensional (3D) imaging of live cells at the submicron scale with high specificity [1]. The advent of the visible fluorescent proteins [2] and of a myriad of fluorescent tags pushed fluorescence microscopy to become the most popular imaging tool in cell biology. The confocal and multiphoton versions of fluorescence microscopy reinforce this condition. In general, is a well-known paradigm the given inability of a lens-based optical microscope to discern details that are closer together than half of the wavelength of light. Recently, the viewpoint for improving resolution moved from optical solutions to the side of the fluorescent molecule to be detected. Today, for the most popular imaging mode in optical microscopy, i.e. fluorescence, the diffraction barrier is crumbling and the term “optical nanoscopy”, coined earlier, comes to be a real far field optical microscope available for the scientific community as the ones allowing individual molecule localization at high precision [3, 4]. Here we discuss about architectures, calibrations and applications of targeted and stochastic readout methods using both single and multiphoton excitation with emphasis towards three-dimensional imaging with high spatial and temporal resolution [5–7].