Andrey S. Klymchenko

Fluorescent probes for sensing fundamental properties of biomolecular environment, such as polarity and hydration, help to study assembly of lipids into biomembranes, sensing interactions of biomolecules and imaging physiological state of the cells. Here, we summarize major efforts in the development of probes based on two photophysical mechanisms: (i) an excited‐state intramolecular charge transfer (ICT), which is represented by fluorescent solvatochromic dyes that shift their emission band maximum as a function of environment polarity and hydration; (ii) excited‐state intramolecular proton transfer (ESIPT), with particular focus on 5‐membered cyclic systems, represented by 3‐hydroxyflavones, because they exhibit dual emission sensitive to the environment. For both ICT and ESIPT dyes, the design of the probes and their biological applications are summarized. Thus, dyes bearing amphiphilic anchors target lipid membranes and report their lipid organization, while targeting ligands direct them to specific organelles for sensing their local environment. The labels, amino acid and nucleic acid analogues inserted into biomolecules enable monitoring their interactions with membranes, proteins and nucleic acids. While ICT probes are relatively simple and robust environment‐sensitive probes, ESIPT probes feature high information content due their dual emission. They constitute a powerful toolbox for addressing multitude of biological questions.