Capturing life (mis)regulation at the nanoscale is a crucial challenge for present and future biophysics. At this scale, the main actors are the molecules. To successfully tackle molecular behavior within living matter, two crucial requisites are typically needed: (i) high spatial resolution (e.g. nanometer), (ii) high temporal resolution (e.g. micro-to-millisecond).

In this context, optical microscopy is a valuable methodological platform: by using fluorescence as signal, spatial and temporal details of molecular processes can be investigated directly within living matter. A number of imaging methods based on fluorescence readouts (e.g. fluorescence fluctuations or lifetime) are currently used to increase the amount of quantitative information that can be extracted from optical microscopy measurements. The multimodal optical signatures collected are converging into the construction of a database of unprecedented complexity.

This in turn stimulates continuous efforts in the development and implementation of new deep-learning algorithms for data handling, analysis, and interpretation. If successful, these new tools are expected to open new perspectives in molecular biophysics and related fields, nanomedicine above all. To photograph the state of the art, a series of applications of advanced optical-microscopy and data-analysis tools to open issues of biological/biomedical interest will be presented and discussed.