ve probes is BODIPY-C₁₀. Yet despite its regular use, BODIPY-C₁₀ remains insufficiently investigated. In this work, we explored how the polarity, hydrogen bonding abilities of the solvent and the presence of macromolecules affect the viscosity-sensing qualities of BODIPY-C₁₀. In addition, we investigated the relaxation pathway of BODIPY-C₁₀ with the help of femtosecond transient absorption and time-dependent DFT calculations. Our results show that while BODIPY-C₁₀ is not affected by protic solvents, accurate quantitative determination of viscosity is possible only if BODIPY-C₁₀ is calibrated in the same polarity environment as in the sample of interest, and the size of the surrounding molecules is not larger than the size of BODIPY-C₁₀. The latter limitation is likely to apply to all molecular rotors.22:T501,Viscosity is a key characteristic of lipid membranes - it governs the passive diffusion of solutes and affects the lipid raft formation and membrane fluidity. Precise determination of viscosity values in biological systems is of great interest and viscosity-sensitive fluorescent probes offer a convenient solution for this task. In this work we present a novel membrane-targeting and water-soluble viscosity probe BODIPY-PM, which is based on one of the most frequently used probes BODIPY-C₁₀. Despite its regular use, BODIPY-C₁₀ suffers from poor integration into liquid-ordered lipid phases and lack of water solubility. Here, we investigate the photophysical characteristics of BODIPY-PM and demonstrate that solvent polarity only slightly affects the viscosity-sensing qualities of BODIPY-PM. In addition, with fluorescence lifetime imaging microscopy (FLIM), we imaged microviscosity in complex biological systems - large unilamellar vesicles (LUVs), tethered bilayer membranes (tBLMs) and live lung cancer cells. Our study showcases that BODIPY-PM preferentially stains the plasma membranes of live cells, equally well partitions into both liquid-ordered and liquid-disordered phases and reliably distinguishes lipid phase separation in tBLMs and LUVs.23:T48a,Lipid droplets (LDs) are cytoplasmic lipid-rich organelles with important roles in lipid storage and metabolism, cell signaling and membrane biosynthesis. Additionally, multiple diseases, such as obesity, fatty liver, cardiovascular diseases and cancer, are related to the metabolic disorders of LDs. In various cancer cells, LD accumulation is associated with resistance to cell death, reduced effectiveness of chemotherapeutic drugs, and increased proliferation and aggressiveness. In this work, we present a new viscosity-sensitive, green-emitting BODIPY probe capable of distinguishing between ordered and disordered lipid phases and selectively internalising into LDs of live cells. Through the use of fluorescence lifetime imaging microscopy (FLIM), we demonstrate that LDs in live cancer (A549) and non-cancer (HEK 293T) cells have vastly different microviscosities. Additionally, we quantify the microviscosity changes in LDs under the influence of DNA-damaging chemotherapy drugs doxorubicin and etoposide. Finally, we show that doxorubicin and etoposide have different effects on the microviscosities of LDs in chemotherapy-resistant A549 cancer cells.

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