Novel coherent multiphoton microscopy of living cells with nanodiamonds

The purpose of this research is to develop a new imaging modality which overcomes a number of severe limitations in currently available methods to observe living cells.
Optical microscopy is an indispensable tool in cell biology, and continuous effort is devoted to develop new techniques with improved performances. Two main approaches can be distinguished: Label-free microscopy techniques versus imaging methods which rely on optical labels. Label-free techniques have the major advantage of looking at unstained cellular and subcellular structures without unwanted artefacts from the labelling procedure. Coherent Antistokes Raman Scattering (CARS) has recently emerged as a powerful label-free method to distinguish biomolecules based on their intrinsic molecular vibrations. However, the benefit of CARS relies on the constructive interference from a large number of identical bonds, hence so far has been mostly successful in distinguishing concentrated lipids in living cells. In order to visualise proteins and DNA with high sensitivity, specificity and at speeds compatible with live cell imaging, optical-labelling is still the only option. In this respect, the most widely utilised labels are fluorescent organic dyes or fluorescent proteins. However, all organic fluorophores are prone to photo-bleaching, an irreversible photo-chemical degradation process severely limiting long time course observations and accompanied by cell toxicity effects.
Alternative to organic fluorophores, solid state inorganic nanoparticles hold a great promise as optical labels in the quest for superior photostability and reduced toxicity. Recently, nanodiamonds (NDs) have gained world-wide attention due to their inexpensive large scale synthesis based on the detonation of carbon containing explosives. They offer particle sizes down to few nm, high biocompatibility and low cytotoxicity and the simple and versatile surface bioconjugation of organic chemistry while keeping the structural integrity of diamond. Their application in optical microscopy of living cells is still at an early stage, with most promising results having been obtained from the fluorescence emission of nitrogen vacancy (NV) centres in diamond. This method is however limited by the efficiency and costs in producing NV centres in NDs. Reports so far have shown that small (<20nm) NDs have a very low probability to have even a single NV center, and that NV centres close to the ND surface are not stable.
In this project, we propose a pilot study to develop a novel way of imaging nanodiamonds in cells which does not rely on (and hence is not limited by) their fluorescence properties. The method is based on the coherent nonlinear light-matter interaction response of NDs and has the added benefit of a superior three-dimensional spatial resolution owing to the nonlinearity of the response. We will explore two types of coherent nonlinearities of NDs: electronically resonant four-wave mixing (FWM) and vibrationally resonant CARS of diamond. The long term vision is the realisation of a new imaging technology that will tackle biological and biomedical problems virtually impossible to address with currently available techniques. As an example, we will follow quantitatively the coherent optical signal of nanodiamonds over time after being internalised in living cells. Our ability to quantify the number of NDs within the cell over time based on the optical signal strength without photobleaching will be a key tool in the study of complicated intracellular pathways.

Our purpose is to develop a new imaging modality to enable the observation of living cells with a superior combination of photostability, absence of phototoxicity, high three-dimensional (3D) spatial resolution and molecular specificity. The technique will be based on diamond nanoparticles (nanodiamonds) as optical labels for cellular imaging which are visualised in a novel way via coherent nonlinear light-matter interaction effects, namely electronically resonant Four-Wave Mixing (FWM) and vibrationally resonant Coherent Antistokes Raman Scatering (CARS). They have not yet been explored experimentally on diamond, hence the need for this pilot study. The long term vision is the realisation of a new imaging technology that will tackle biological and biomedical problems virtually impossible to address with currently available techniques. As an example, we will show the superior ability of the technique to follow quantitatively the coherent optical signal of nanodiamonds over time after being internalised in living cells, in order to decipher the trafficking of molecules through complicated endocytic intracellular pathways.

Who will benefit from this research?

The imaging modality developed in this pilot study will progress the field of optical microscopy applied to cell biology and will advance our understanding of the interaction between cells and nanoparticles. Hence this research will impact:
i) The academic community working in a wide range of disciplines including optical engineering, physics, chemistry, material science, biology and medicine (see Academic beneficiaries section).
ii) The commercial sector, through microscope manufacturers and laser companies interested in the exploitation of the FWM and CARS technology, but also drug discovery companies interested in the visualization and tracking of nanodiamonds in cells as novel bio-compatible carriers for drug delivery.
iii) The public sector, through the benefit for public health in the development of novel therapeutic strategies.

How will they benefit from this research?

The contribution of this research to these beneficiaries and to the nation's health, wealth and culture will be mainly through:
- Knowledge: via the scientific advancements (on a 1-3 years realistic timescale) in the microscopy technology and its biological applications.
- People: The postdoctoral RA on this project will receive state of the art training in biophotonics. In 2006, Cardiff University established the first MSc Biophotonics course in the UK, jointly taught between the School of Physics and the School of Biosciences, sponsored by microscopy-related industrial collaborators. Three of the applicants teach and/or organize modules on this course. The microscopy modality developed in this project will add to the depth of the research training offered within the course, and will allow better recruiting of excellent students to be trained as Biophotonics leaders of tomorrow.
- Improvement of health and quality of life. The direct research outputs will lead to greater understanding of membrane trafficking and endocytosis in living cells which play a key role in many viral diseases and in drug delivery. Translation of these research outputs into therapeutic strategies will ultimately benefit public health (realistic timescale > 10years).

What will be done to ensure that they benefit from this research?

The research team in this programme will undertake impact activities as detailed in the Pathways to Impact. Briefly, the team will engage with secondary Schools as STEM ambassadors. Communication with industry and exploitation will occur through an Imaging Symposium annual event, a KTP showcase event and via IP protection routes. The PI has a track record in the organisation of Biophotonics conferences at UK and international level. Within the time frame of this project, she will organize and chair the Biophotonics session at the Photon 2012 event in Durham, UK. Photon12 is the largest optics conference in the UK, and the outcome of this project will be showcased at this event. Within the School of Biosciences, specialised staff have been appointed in the form of Innovation and Engagement Officers as detailed in the Pathways to Impact.