The application of Ru(II) polypyridyl complexes to cellular imaging and sensing

Fluorescent microscopy is the key bio-imaging tool that is used to study live cells. Luminescent transition metal complex have been explored extensively for many years across a range of applications from solar energy to molecular therapeutics, but it is only over the last decade that they have been seriously considered as cellular imaging probes. Their unique photophysical properties including large Stokes shift, red emission wavelengths, good photostability, and sensitivity to molecular oxygen mean they are more than just contrast agents, making them invaluable in diagnostics and theranostics. A key aim of this thesis was to drive forward the demonstrated applications of luminescent Ru(II) and Os(II) probes conjugated to cell penetrating peptides in cellular imaging and sensing using confocal microscopy, and the relatively new phenomenon of super resolution microscopy, specifically, STimulated Emission Depletion (STED) microscopy. Chapter 1 overviews the general photophysical and optical of ruthenium and osmium polypyridyl complexes, examining the current status of their application in live cellular imaging and sensing. Microscopy techniques such as confocal laser scanning microscopy, stimulated emission depletion (STED) microscopy and fluorescence lifetime imaging microscopy (FLIM) are discussed and how they are used alongside luminescent probes for cellular sensing. Despite its NIR emission and high photostability Os(II) complexes have not been reported as general imaging probes before this report. Chapter 2 describes a novel Os(II) complex conjugated to the octa-arginine (R8) cell-penetrating peptide sequence as a cellular imaging probe, and compares it to its Ru(II) analogue. The photophysical properties of [Os(bpy)2(pic-arg8)]10+ and [Ru(bpy)2(pic-arg8)]10+ are examined and compared, as well as its ability to be taken up by live CHO and Sp2 cells, examined by confocal microscopy. Chapter 3 outlines a novel Ru(II) oxygen sensing probe, conjugated to the mitochondrial localising peptide sequence FrFkFrFk, in order to direct the complex to the mitochondria of live HeLa cells using confocal microscopy to confirm location. Fluorescent lifetime imaging microscopy (FLIM) allowed [(Ru(bpy)2phen-Ar)2-FrFKFrFK]7+ to measure oxygen concentrations and reactive oxygen species (ROS) from within the mitochondria of live HeLa cells. Chapter 4 focuses on ratiometric oxygen sensing in live cells. Here, two probes are examined – a ratiometric molecule and a ratiometric core-shell nanoparticle. In both cases, the oxygen-sensitive probe consists of a Ru(II) complex, while the oxygeninsensitive reference probe is a BODIPY complex. Finally, Chapter 5 describes the application of Ru(II) probes to STimulated Emission Depletion (STED) microscopy. Here, signal peptides conjugated to two novel Ru(II) probes directs them to the endoplasmic reticulum (ER) and nucleus of HeLa cells. The probes performance under confocal microscopy and STED is compared in the improved image resolution achieved as well as their photostability under the intense STED xii depletion laser. High resolution images of the ER and nuclear DNA during the stages of mitosis are captured using STED microscopy