1. The search of most optimal fluorescent probes
The implementation of this project requires the use of fluorescent probes for viscosity and polarity for systematic work with animal (porcine) and human eye lenses. A set of available molecular rotors including newly sensitized fluorescent dyes (in total 18 molecules) was tested for their localisation within plasma membranes, the signal intensity, and the functioning as viscosity reporters. Based on this screening two molecules were chosen as the major molecular rotors for this project. Viscosity calibration curves were revised with the purpose to extend the existing calibration to high viscosity region, 2000 – 12 000 cP, a significant improvement on previous studies. One of the newly reported polarity probes (pyrene-based probe, PK) has revealed a bright signal and good localisation within plasma membranes for both young porcine and aged human lenses.
2. Photodamage to plasma membranes of eye lens
The Solar UV-A radiation (315-400 nm) on the Earth’s surface is considered as the most important external source of modifications of proteins and lipids within the eye lens. Proteins are currently considered to be the major targets for photodamage, and little is known about photodamage to plasma membranes.
In this work the impact of different photoreactions via electron / proton transfer (Type I) or singlet oxygen (Type II) on the plasma membranes of eye lens was studied with porcine eye lenses. We demonstrated that these two types of photodamage result in clearly distinct changes in viscosity – a decrease in the case of Type I damage and an increase in the case of Type II processes. At the final step of this study, we simulated age-related changes that occur in vivo via an exposure of an intact eye lens to UV-A light under anaerobic conditions. The observed decrease in viscosity within plasma membranes is consistent with the ability of eye lens constituents to sensitize Type I photodamage under natural irradiation conditions. These changes are likely to alter the transport of metabolites and predispose the whole tissue to the development of pathological processes such as cataracts. These results were published in a full paper in J. Photochem. Photobiol. B: Biology.
3. Age-related changes in viscosity of plasma membranes of human eye lenses
Experiments with non-fixed human lenses revealed significant differences in the morphology of cells as compared with fixed tissues in previously published histology works. Three types of cell morphologies have been revealed by our studies, with clear distinction between different types of cells. Polarity and viscosity profiles were recorded across slices giving unexpected results in the observed viscosities. The correct interpretation of these results requires the accumulation of further statistics as well as the measurement of viscosities within cytosol. Unfortunately, the schedule of this work was affected by the COVID-19 pandemic and restrictions, primarily affecting the delivery of human samples.
Exploitation and dissemination
The microscopy work with lens tissue has not been attempted previously, yielding many new protocols in procedures for sample preparation, acquisition and analysis of data. The main outcomes of this project are (i) data used by other lab members working with biological tissues, (ii) new directions of further research involving ocular tissues, (iii) publications resulting from this work.
Due to COVID-19 induced restrictions to mobility, the results were mainly disseminated via group seminars. The Fellow participated in one on-line meeting organised by RSC Photophysics and Photochemistry Group with the delivery of results as a pre-recorded video. Another important channel of dissemination was organising new collaborations and research meetings with groups of Prof M. Wormstone (University of East Anglia, the UK) and Prof Y. Rotenstreich (Sheba Medical Centre, Israel), which expressed large interest in the application of FLIM method in their eye-related studies.
Personal development
At Imperial College I received training on microscopy, fluorescence imaging and work with live cells, which were used to highlight differences in structures between live cells and fibrous lens cells. From my side, I transferred my knowledge in the work with ocular tissues and their preparations for microscopy and different ways of photodamage to the whole or sliced tissues.
