Viscosity can be the major factor governing a system's bulk mechanical properties and internal processes on the microscale such as molecular diffusion. Mechanical and rheological properties measured on the microscale may differ widely from the macroscopic properties of a physical system. While they are of extreme interest in biology, chemistry and in natural sciences, these microscopic properties are often very challenging to measure due to small dimensions of the system under study. Lifetime-based molecular rotors, fluorescent molecules whose fluorescence lifetime decay profile is sensitive to the microviscosity of their environment, can be used in Fluorescence Lifetime Imaging (FLIM) to acquire lifetime/viscosity images of high spatial and temporal resolution. In our study we used molecular rotors to overcome the inherent limitations of current mechanical and spectroscopic methods and to measure and image microviscosity in: i) cells during oxidative stress from singlet oxygen during Photodynamic Therapy (PDT) and ii) organic aerosol particles during chemical ageing and hydration.

Organic aerosols (OA) play a prominent role in many atmospheric processes, climate change and can affect human health. Their physical and chemical properties control their lifetime, reaction rates and optical properties. In our study, we utilised a rare combination of optical trapping and FLIM to measure OA viscosity changes, in their true aerosol phase, during atmospherically-relevant oxidation with ozone and hydroxyl radicals. The results were compared with oxidation experiments of OA deposited on the flat surface of a coverslip. The well established lifetime-based rotor Bodipy-C10 was used as a hydrophobic rotor during oxidation of model OA squalene particles. Our results showed a fast viscosity increase to the point of solidification and revealed the development of spatial viscosity heterogeneities during oxidation.

A new hydrophilic molecular rotor, thiazole orange (TO), was characterized and used to measure secondary organic aerosol (SOA) viscosity under different relative humidity (RH) conditions, along with the already well characterised lifetime-based hydrophilic rotor Cy3. TO fluorescence quantum yield and lifetime profile showed a marked sensitivity in the high range of viscosities, unlike any other known rotor, rendering TO an ideal viscosity sensor for the highly viscous SOA particles. Atmospherically relevant SOA particles derived from three different volatile compounds (VOCs) were measured and their viscosity was found to vary from hundreds to millions centipoise. The dynamic changes in viscosity of SOA were also measured during a sudden RH change.

Intracellular viscosity was measured in the hydrophobic areas of live monolayers of cells using Bodipy-C10 during PDT with two different photosensitisers. Such measurement where the photosensitiser that induced the treatment and the rotor were separate molecules was performed for the first time. Consequently, we were able to use the same rotor (Bodipy-C10) and two different photosensitisers (PSs) with different intracellular localisation: verteporfin (VP) and porphycene (PO1). Their different localisation in cells was confirmed by confocal microscopy. Our results showed a marked viscosity increase over irradiation with both PSs, which resulted in singlet oxygen production and cell death as a result of PDT. This viscosity increase was observed both in the irradiated intracellular areas but also at distant non irradiated locations, believed to result from the formation and diffusion of long lived peroxides. The highest viscosity values measured were different for VP and PO1, as was the distance of remote damage. Overall, the potential of molecular rotors as viscosity sensors was demonstrated in biological and physical systems, providing unprecedented insight on the mechanical properties, with the emphasis on monitoring dynamic changes in viscosity. Our results revealed additional levels of complexity in the systems under study, which was not possible to observe using any other currently available techniques for monitoring viscosity.