Many biological processes are based on chemical reactions. Viscosity determines how fast molecules can diffuse, and react. Therefore in cells viscosity can affect signalling, transport and drug delivery, and abnormal viscosity has been linked to disease and malfunction. In spite of its importance, measuring viscosity on a scale of a single cell is a challenge. Traditionally used mechanical methods are no longer applicable and must be substituted by a spectroscopic approach. Such spectroscopic approaches exist, e.g. single particle tracking, monitoring the rate of fluorescence recovery after photobleaching, or monitoring the rate of viscosity-dependent photochemical reactions. However all of the above are single point measurements and in a complex heterogeneous environment of a cell can not provide full information. The spectroscopic approach which allows imaging or mapping of viscosity would be of great benefit. This proposal aims to measure and map viscosity inside a single cell with high precision and high spatial resolution using novel fluorescent probes, called molecular rotors. In molecular rotors fluorescence competes with intramolecular rotation. In a viscous environment rotation is slowed down and this strongly affects fluorescence. Thus viscosity can be measured by detecting the change in either the fluorescence spectra or lifetimes. Existing technology allows imaging of either the fluorescent spectra or lifetimes with excellent spatial resolution in single live cells. To date we have produced maps of viscosity in certain parts of cells using this approach and demonstrated that local viscosity in those compartments can be up to 100x higher than that of water.Important advantage of molecular rotor approach is a very short measurement time. Using this advantage, this proposal aims to monitor how viscosity in a cell changes during dynamic biological processes, e.g. change in the membrane structure upon cell perturbation, drug administration and cell death.Photodynamic therapy (PDT) is a form of cancer treatment, which relies on the generation of short-lived toxic agents within a cell upon irradiation of a drug. The efficacy of this treatment critically depends on the viscosity of the medium through which the cytotoxic agent must diffuse during its short life span. This proposal will monitor how cell viscosity and other vital biophysical cell parameters change during PDT. The novelty of our approach is in using spatially resolved irradiation of the drug within cells. E.g. we can irradiate a single organelle and monitor the change in the entire cell. Alternatively, we can irradiate the group of cells and monitor the behaviour of its neighbours. This approach is ideal tool to directly probe the 'bystander effect', when the cells which have not been directly treated show significant response to therapy, the effect which is very important in radiation and PDT cancer treatment. This proposal will be carried out in the Chemistry Department at Imperial College London where multidisciplinary collaborations are established to ensure the success of the work proposed. This project will address both the fundamental scientific issues in photochemistry and cell biology and also encourage the development of applications, such as measuring viscosity as a diagnostic tool and for monitoring the progress of treatments.