Shedding new light on cells with coherent multiphoton nanoscopy

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The aim of this research is the realization of a novel imaging modality to enable the observation of living cells and tissues under physiological conditions with unprecedented sensitivity and spatial resolution, without the need to stain them with fluorophores. The technique, based on the interaction of light with matter in the coherent regime, will feature a unique combination two process: Coherent Antistokes Raman Scattering (CARS) of biomolecules in living cells and Four-Wave Mixing (FWM) imaging of metallic nanoparticles (NPs). This technology will progress the field of optical 'nanoscopy', advance our understanding in physics and material sciences, tackle biological problems that are virtually impossible to address with currently available techniques, and will be of relevance in medical applications to improve the diagnostic and treatment of diseases.Optical microscopy is an indispensable tool that is driving progress in cell biology, however most cellular constituents have no colour and they are hard to distinguish under a light microscope unless they are stained. Fluorescence microscopy using organic dyes attached to biomolecules or fluorescent proteins has provided a highly sensitive method of visualizing biomolecules. However, when used for observations in living cells, these modified biomolecules raise questions if their behaviour is real or artefactual. Furthermore, all organic fluorophores are prone to photo-bleaching, an irreversible degradation of the fluorescence intensity after excitation with light, which severely limits time-course observations and is accompanied by toxicity effects and consequent cell damage. In CARS the image contrast is obtained by detecting light which is scattered by vibrating bonds in unstained biomolecules. Although this scattering phenomenon produces a very weak signal, it can be coherently enhanced when two short laser pulses are used to excite the vibrations (generating CARS) so that the scattered light from all bonds of the same type constructively interfere. However, CARS still requires a high number of molecules to achieve sufficient signal for detection, and the existence of a background severely limits its sensitivity. Another problem is the spatial resolution limited by the optical diffraction (>100nm).In this programme, I will overcome these limitations by developing a background free CARS detection combined with the light enhancement occurring in the nanoscale range near a metallic NP, to achieve nanometric spatial resolution and high sensitivity. The NP will be located and tracked using FWM imaging, recently demonstrated in our laboratory, here in a new version to enable nanometric position accuracy in all three directions. FWM detection will also report the local thermal conductivity of the NP surroundings. In addition the microscope will feature trapping of the NP with optical tweezers to position it in a region of interest and/or to measure forces applied to it.The ability to map the intrinsic chemical composition of nanoscale regions together with their thermal and mechanical properties in living cells will have a major impact in solving important biomedical problems. For example, we will determine whether cell membranes perform their function through the assembly of lipid nanodomains or 'rafts'. Their existence is thought to play a key role in basic biological functions and in many diseases (eg influenza and HIV) but is also controversial owing to their small size. Another application will be to determine the local membrane environment associated with endocytosis which is crucial, beyond fundamental biology, for the design of drug delivery and therapeutic strategies. More in general, this novel imaging modality will allow us to address biological systems where the manipulation and photo-toxicity associated with the use of fluorescence markers is unacceptable, e.g. in the areas of in-vitro fertilization and cancer research.