Confocal and Two-Photon Spectroscopy

The spectroscopic analysis of bulk samples provides useful information at the mesoscopic scale that can be fostered when coupled with microscopy, which allows to integrate the spatial information in the analysis, this being of utmost importance for non‐homogeneous samples. Technologies such as confocal and two‐photon microscopy quickly became mainstream methods as they provide the possibility of noninvasive real‐time analysis of specimens of different nature, eliciting specific features even in samples with non‐ergodic behaviour. The spatial resolution of optical microscopy has its roots in the diffraction of light on the objective lens of the imaging system. Fluorescence microscopy is an optical microscopy technique in which the detected signal arises from the fluorescent molecules of the analyzed materials. Modern confocal and multiphoton microscopes allow direct investigation of fluorescent molecules/ structures in a spatially resolved manner. Fluorescence microscopy allows a plethora of spectroscopic methods, which give the opportunity to characterize molecules and interactions within the nanometer scale.