Reactive Oxygen Species (ROS) cause DNA damage and defective function in sperm and also affects the developmental competence of embryos. It is therefore critical to monitor ROS in sperm, oocytes and developing embryos. In particular, hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) is a ROS important to normal cell function and signalling as well as its role in oxidative stress. Here we report the development of a fluorescent sensor for H<sub>2</sub>O<sub>2</sub> using carboxyperoxyfluor-1 (CPF1) in solution and attached to a glass slide or multi-mode optical fibre. CPF1 increases in fluorescence upon reaction with H<sub>2</sub>O<sub>2</sub> to non-invasively detect H<sub>2</sub>O<sub>2</sub> near developing embryos. These probes are constructed by immobilising CPF1 to the optical fibre tip a polyacrylamide layer. Also reported is a new dual optical fibre sensor for detecting both H<sub>2</sub>O<sub>2</sub> and pH that is functional at biologically concentrations of H<sub>2</sub>O<sub>2</sub> and can sense pH to 0.1 units. This research shows promise for the use of optical fibre sensors for monitoring the health of developing embryos. Furthermore, these sensors are applicable for use beyond embryos such as detecting stress in endothelial cells involved in cardiovascular dysfunction.
Automated and unbiased methods of non-invasive cell monitoring able to deal with complex biological heterogeneity are fundamentally important for biology and medicine. Label-free cell imaging provides information about endogenous fluorescent metabolites, enzymes and cofactors in cells. However extracting high content information from imaging of native fluorescence has been hitherto impossible. Here, we quantitatively characterise cell populations in different tissue types, live or fixed, by using novel image processing and a simple multispectral upgrade of a wide-field fluorescence microscope. Multispectral intrinsic fluorescence imaging was applied to patient olfactory neurosphere-derived cells, cell model of a human metabolic disease MELAS (mitochondrial myopathy, encephalomyopathy, lactic acidosis, stroke-like syndrome). By using an endogenous source of contrast, subtle metabolic variations have been detected between living cells in their full morphological context which made it possible to distinguish healthy from diseased cells before and after therapy. Cellular maps of native fluorophores, flavins, bound and free NADH and retinoids unveiled subtle metabolic signatures and helped uncover significant cell subpopulations, in particular a subpopulation with compromised mitochondrial function. The versatility of our method is further illustrated by detecting genetic mutations in cancer, non-invasive monitoring of CD90 expression, label-free tracking of stem cell differentiation, identifying stem cell subpopulations with varying functional characteristics, tissue diagnostics in diabetes, and assessing the condition of preimplantation embryos. Our optimal discrimination approach enables statistical hypothesis testing and intuitive visualisations where previously undetectable differences become clearly apparent.
The production of reactive oxygen species (ROS) is known to affect the developmental competence of embryos. Hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>) an important reactive oxygen species, is also known to causes DNA damage and defective sperm function. Current techniques require incubating a developing embryo with an organic fluorophore which is potentially hazardous for the embryo. What we need is a localised ROS sensor which does not require fluorophores in solution and hence will allow continuous monitoring of H<sub>2</sub>O<sub>2</sub> production without adversely affect the development of the embryo. Here we report studies on such a fibre-based sensor for the detection of H<sub>2</sub>O<sub>2</sub> that uses a surface-bound aryl boronate fluorophore carboxyperoxyfluor-1(CPF1). Optical fibres present a unique platform due to desirable characteristics as dip sensors in biological solutions. Attempts to functionalise the fibre tips using polyelectrolyte layers and (3-aminopropyl)triethoxysilane (APTES) coatings resulted in a limited signal and poor fluorescent response to H<sub>2</sub>O<sub>2</sub> due to a low tip surface density of the fluorophore. To increase the surface density, CPF1 was integrated into a polymer matrix formed on the fibre tip by a UV-catalysed polymerisation process of acrylamide onto a methacrylate silane layer. The polyacrylamide containing CPF1 gave a much higher surface density than previous surface attachment methods and the sensor was found to effectively detect H<sub>2</sub>O<sub>2</sub>. Using this method, biologically relevant concentrations of H<sub>2</sub>O<sub>2</sub> were detected, enabling remote sensing studies into ROS releases from embryos throughout early development.