High-resolution optical imaging techniques make now accessible the detection of nanofeatures in bio- and soft-matter by non-ionizing visible radiation. However, high-resolution imaging is critically dependent by the fluorescent probes used for reporting on the nano-environment. On account of our long-standing interest in the development of fluorescent probes, we set out to design and engineer newfluorescent systems for nanoscale imaging and sensing of biological specimens and soft-matter. These fluorophores report on fast subtle changes of their nanoscale environmentat excited state and are meant to fulfill these requirements: a) optical responses (intensity, wavelength-shift, lifetime, anisotropy) predictably related to the environmental polarity, viscosity, macromolecular structure, b) high brightness allowing for single-molecule detection, c) easily conjugable to biomolecules or macromolecules of interest. Notably, we aim at conjugating these properties with the capability of nanoscopy imaging based on Stimulated Emission Depletion or Stochastic Reconstruction Optical Microscopy. In this lecture the main features and applications of the engineered probes will be reviewed and future developments in this exciting field will be discussed.
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