Dr Joshua Hughes

In drug design, understanding the subcellular localisation and physicochemical behaviour of candidate molecules is essential for optimising their efficacy and elucidating their mechanisms of action. Imaging probes are routinely employed for this purpose, though most studies rely on a single imaging modality. By integrating multiple imaging techniques into a multimodal system, a more comprehensive understanding of localisation, microenvironment, and physicochemical interactions can be achieved. Yet, there remains a scarcity of specialised imaging probes with well-defined photophysical profiles, especially those that combine luminescence with a Raman-active tag in the cell-silent region, where signal clarity is maximised. Such probes could function independently or be conjugated to drugs or targeting moieties as dual-mode imaging tags. In this study, we showcase the application of a suite of advanced imaging modalities: fluorescence microscopy (CLSM); Fluorescence Lifetime Imaging Microscopy (FLIM); Phosphorescence Lifetime Imaging Microscopy (PLIM); and simultaneous fluorescence and Raman spectroscopy (FluoRaman). The modalities were exemplified by a series of novel, highly solvatochromic diarylacetylene-based photosensitisers that feature alkyne Raman tags and exhibit diverse subcellular localisation.

Reactive oxygen species (ROS) are naturally produced compounds that play important roles in cell signaling, gene regulation, and biological
defense, including involvement in the oxidative burst that is central to the anti-microbial actions of macrophages. However, these highly
reactive, short-lived radical species also stimulate cells to undergo programmed cell death at high concentrations, as well as causing
detrimental effects such as oxidation of macromolecules at more moderate levels. Imaging ROS is highly challenging, with many researchers
working on the challenge over the past 10–15 years without producing a definitive method. We report a new fluorescence microscopy-based
technique, Bullseye Analysis. This methodology is based on concepts provided by the FRAP (Fluorescence Recovery after Photobleaching)
technique and refined to evidence the spatiotemporal production of ROS, and the subsequent consequences, on a subcellular scale. To
exemplify the technique, we have used the ROS-reporter dye, CellROX, and the ROS-inducing photosensitizer, LightOx58, a potent source of
ROS compared with UV irradiation alone. Further validation of the technique was carried out using differing co-stains, notably Mitotracker and
JC-1.

Fluorescent probes are increasingly used as reporter molecules in a wide variety of biophysical experiments, but when designing new compounds it can often be difficult to anticipate the effect that changing chemical structure can have on cellular localisation and fluorescence behaviour. To provide further chemical rationale for probe design, a series of donor–acceptor diphenylacetylene fluorophores with varying lipophilicities and structures were synthesised and analysed in human epidermal cells using a range of cellular imaging techniques. These experiments showed that, within this family, the greatest determinants of cellular localisation were overall lipophilicity and the presence of ionisable groups. Indeed, compounds with high logD values (>5) were found to localise in lipid droplets, but conversion of their ester acceptor groups to the corresponding carboxylic acids caused a pronounced shift to localisation in the endoplasmic reticulum. Mildly lipophilic compounds (logD = 2–3) with strongly basic amine groups were shown to be confined to lysosomes i.e. an acidic cellular compartment, but sequestering this positively charged motif as an amide resulted in a significant change to cytoplasmic and membrane localisation. Finally, specific organelles including the mitochondria could be targeted by incorporating groups such as a triphenylphosphonium moiety. Taken together, this account illustrates a range of guiding principles that can inform the design of other fluorescent molecules but, moreover, has demonstrated that many of these diphenylacetylenes have significant utility as probes in a range of cellular imaging studies.

The emergence of antibiotic resistance is a growing threat to human health, and therefore, alternatives to existing compounds are urgently needed. In this context, a novel fluorescent photoactivatable diarylacetylene has been identified and characterised for its antibacterial activity, which preferentially eliminates Gram-positive over Gram-negative bacteria. Experiments confirmed that the Gram-negative lipopolysaccharide-rich outer surface is responsible for tolerance, as strains with reduced outer membrane integrity showed increased susceptibility. Additionally, bacteria deficient in oxidative damage repair pathways also displayed enhanced sensitivity, confirming that reactive oxygen species production is the mechanism of antibacterial activity. This new diarylacetylene shows promise as an antibacterial agent against Gram-positive bacteria that can be activated in situ, potentially for the treatment of skin infections.