Cellular ROS imaging with hydro-Cy3 dye is strongly influenced by mitochondrial membrane potential

Background: Hydrocyanines are widely used as fluorogenic probes to monitor reactive oxygen species (ROS) generation in cells. Their brightness, stability to autoxidation and photobleaching, large signal change upon oxidation, pH independence and red/near infrared emission are particularly attractive for imaging ROS in live tissue. Methods: Using confocal fluorescence microscopy we have examined an interference of mitochondrial membrane potential (ΔΨm) with fluorescence intensity and localisation of a commercial hydro-Cy3 probe in respiring and non-respiring colon carcinoma HCT116 cells. Results: We found that the oxidised (fluorescent) form of hydro-Cy3 is highly homologous to the common ΔΨm-sensitive probe JC-1, which accumulates and aggregates only in ‘energised’ negatively charged mitochondrial matrix. Therefore, hydro-Cy3 oxidised by hydroxyl and superoxide radicals tends to accumulate in mitochondrial matrix, but dissipates and loses brightness as soon as ΔΨm is compromised. Experiments with mitochondrial inhibitor oligomycin and uncoupler FCCP, as well as a common ROS producer paraquat demonstrated that signals of the oxidised hydro-Cy3 probe rapidly and strongly decrease upon mitochondrial depolarisation, regardless of the rate of cellular ROS production. Conclusions: While analysing ROS-derived fluorescence of commercial hydrocyanine probes, an accurate control of ΔΨm is required. General significance: If not accounted for, non-specific effect of mitochondrial polarisation state on the behaviour of oxidised hydrocyanines can cause artefacts and data misinterpretation in ROS studies.

Development and assessment of new sensor systems in food packaging applications

The use of optical sensor technology for non-invasive determination of key quality pack parameters improved package/product quality. This technology can be used for optimization of packaging processes, improvement of product shelf-life and maintenance of quality. In recent years, there has been a major focus on O2 and CO2 sensor development as these are key gases used in modified atmosphere packaging (MAP) of food. The first and second experimental chapters (chapter 2 and 3) describe the development of O2, pH and CO2 solid state sensors and its (potential) use for food packaging applications. A dual-analyte sensor for dissolved O2 and pH with one bi-functional reporter dye (meso-substituted Pd- or Ptporphyrin) embedded in plasticized PVC membrane was developed in chapter 2. The developed CO2 sensor in chapter 3 was comprised of a phosphorescent reporter dye Pt(II)- tetrakis(pentafluorophenyl) porphyrin (PtTFPP) and a colourimetric pH indicator α-naphtholphthalein (NP) incorporated in a plastic matrix together with a phase transfer agent tetraoctyl- or cetyltrimethylammonium hydroxide (TOA-OH or CTA-OH). The third experimental chapter, chapter 4, described the development of liquid O2 sensors for rapid microbiological determination which are important for improvement and assurance of food safety systems. This automated screening assay produced characteristic profiles with a sharp increase in fluorescence above the baseline level at a certain threshold time (TT) which can be correlated with their initial microbial load and was applied to various raw fish and horticultural samples. Chapter 5, the fourth experimental chapter, reported upon the successful application of developed O2 and CO2 sensors for quality assessment of MAP mushrooms during storage for 7 days at 4°C.

An exploration through interview and drawings of the experiences of patients diagnosed with oral cancer across their cancer trajectory

Background: The treatment of oral cancer is complex and lengthy. Curative treatment implies a combination of surgery, radiotherapy and chemotherapy. The main goal of treatment is to guarantee long-term tumour free survival with as little functional and cosmetic damage. Despite progress in developing these strategies, cancers of the oral cavity continue to have high mortality rates that have not improved dramatically over the past ten years. Aim: The aim of this study was to uniquely explore the dynamic changes in the physical, psychological, social and existential experiences of newly diagnosed patients with oral cancer at two points across their cancer illness trajectory i.e. at the time of diagnosis and at the end of treatment. Methodology: A qualitative prospective longitudinal design was employed. Non-probability purposive sampling allowed the recruitment of 10 participants. The principal data collection method used was a digital audio taped semi-structured interview along with drawings produced by the participants. Analysis: Data was analysed using latent content analyses. Summary: Three ‘dynamic’ themes, physical, psychosocial and existential experiences were revealed that interact and influence each other in a complex and compound whole. These experiences are present at different degrees and throughout the entire trajectory of care. Patients have a number of specific concerns and challenges that cannot be compartmentalised into unitary or discrete aspects of their daily lives. Conclusion & Implications: An understanding of the patient’s experience of their illness at all stages of the disease trajectory, is essential to inform service providers’ decision making if the delivery of care is to be client centred. Dynamic and fluctuating changes in the patient’s personal experience of the cancer journey require dynamic, energetic and timely input from health care professionals.

New phosphorescence based probes and techniques for the analysis of cellular oxygen and respiration

Real time monitoring of oxygenation and respiration is on the cutting edge of bioanalysis, including studies of cell metabolism, bioenergetics, mitochondrial function and drug toxicity. This thesis presents the development and evaluation of new luminescent probes and techniques for intracellular O2 sensing and imaging. A new oxygen consumption rate (OCR) platform based on the commercial microfluidic perfusion channel μ-slides compatible with extra- and intracellular O2 sensitive probes, different cell lines and measurement conditions was developed. The design of semi-closed channels allowed cell treatments, multiplexing with other assays and two-fold higher sensitivity to compare with microtiter plate. We compared three common OCR platforms: hermetically sealed quartz cuvettes for absolute OCRs, partially sealed with mineral oil 96-WPs for relative OCRs, and open 96-WPs for local cell oxygenation. Both 96-WP platforms were calibrated against absolute OCR platform with MEF cell line, phosphorescent O2 probe MitoXpress-Intra and time-resolved fluorescence reader. Found correlations allow tracing of cell respiration over time in a high throughput format with the possibility of cell stimulation and of changing measurement conditions. A new multimodal intracellular O2 probe, based on the phosphorescent reporter dye PtTFPP, fluorescent FRET donor and two-photon antennae PFO and cationic nanoparticles RL-100 was described. This probe, called MM2, possesses high brightness, photo- and chemical stability, low toxicity, efficient cell staining and high-resolution intracellular O2 imaging with 2D and 3D cell cultures in intensity, ratiometric and lifetime-based modalities with luminescence readers and FLIM microscopes. Extended range of O2 sensitive probes was designed and studied in order to optimize their spectral characteristics and intracellular targeting, using different NPs materials, delivery vectors, ratiometric pairs and IR dyes. The presented improvements provide useful tool for high sensitive monitoring and imaging of intracellular O2 in different measurement formats with wide range of physiological applications.