Advanced multidimensional optics to investigate biological complexity at the single-molecule level in living, functional cells

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What is the physical, molecular basis of the cell? How do single-molecule mesoscopic properties in a living organism scale up to effect whole-organism functionality? Can we bridge our gap in understanding between the physical characteristics of bio-molecules to the ultimate manifestation of a biological super-computer which is the single cell, in a rational, predictive context? These are the major outstanding challenges to the future of biologically-inspired physics research, but ones which I propose to address by development of radical new technologies. This proposal describes the construction and further development of an exceptionally versatile, ultra-sensitive custom-built microscope capable of monitoring dynamic localization and stoichiometry of functional molecular complexes in living cells to a precision of single molecules in real-time with super-resolution optical performance. Most of the vital activities in living cells are carried out by proteins, so small that 1 billion could fit on a full-stop. Many of these processes require collections of proteins to assemble together into functional biological machines. This proposed microscope will allow us to determine precisely how many components a machine has, how they assemble and disassemble, how they mechanistically interact with each other and their surroundings, how dynamic their molecular stability is and how machines of different biological processes co-operate to produce potent, compounded effects at the level of the whole cell. A full insight into the mechanisms of living cells can be achieved only by investigating the key interactions that elicit and direct cellular events, though to date the shear complexity of biological systems has caused precise single-molecule experimentation to be far too demanding, instead focusing on studies of single systems using relatively crude bulk ensemble-average measurements which use in effect the summed signals from many thousands of molecules. What I now propose is to monitor several biological systems simultaneously in a living functioning cell using more powerful and precise single-molecule techniques, investigating from a bottom-up molecular physics level. But, to achieve this experimentally requires a rigorous and dedicated physics-research approach. The microscope will consist of a laser that can output multiple wavelengths of controllable polarization states which can alternate excitation for a variety of coloured fluorescent-dipole tags attached to several distinct proteins in the same biological sample, with fluorescence light emissions being imaged using high quantum-efficiency thermoelectrically cooled wide-field photon counting technology. Short wavelength excitation will allow controlled photo-activation of modified molecular dyes permitting exceptional super-resolution capability beyond the optical far-field diffraction limit. My optical-physics approach offers a minimally-perturbative, non-invasive means to probe functional living cells, features not readily available to most existing single-molecule approaches of biologically-inspired physics. This will enable the study of molecular function within the true, intact, original biological context. The techniques developed will be of immense interdisciplinary benefit for physicists, biologists, chemists, engineers, mathematicians and computer scientists, with a strong potential for commercial exploitation in bio-sensing and native-cell drug validation for the pharmaceutical industry. Following in vivo imaging optimization on relatively low complexity bacterial cells will pave the way towards more challenging studies on complex multi-cellular systems, culminating in medical research on human cell lines involved in the investigation of disease and in establishing therapeutics.