FROM MICROSCOPY TO NANOSCOPY: HOW TO GET AND READ OPTICAL DATA AT SINGLE MOLECULE LEVEL USING CONFOCAL AND TWO-PHOTON EXCITATION MICROSCOPY.

The application fluorescence to confocal and two-photon excitation (2PE) optical microscopy has led to terrific advances in the study of biological systems from the three-dimensional (3D) micro-spectroscopic level down to single molecule detection (SMD) schemes. Both techniques are particularly relevant for the study of the 3D and dynamic properties of biological molecules within their natural environment, cells or tissues. In particular the advent of 2PE mitigates overall photobleaching and phototoxicity problems, opening new perspectives by providing further attractive advantages. Optical schemes and architectures for confocal and two-photon excitation from microscopic level to SMD will be discussed. Examples of three-dimensional and multiple fluorescence imaging from cells to single fluorescent molecules will be given. Examples in the utilization of confocal and 2PE for specific GFP switching at single molecule level and for monitoring of TPE uncaging will be shown.