Nonlinear plasmonic biosensing and functional imaging

This additional support for EPSRC Research Leaders will be used to fund two interrelated strands of work which synergise with the current Leadership fellowship whilst expanding into new directions. The work will focus on a new avenue of using the coherent optical nonlinearity (called Four-Wave Mixing - FWM) of pairs of metallic nanoparticles (NPs) for biosensing single molecules directly inside cells, and on the combination of FWM with electron microscopy as a new correlative light electron microscopy (CLEM) approach.
For an in depth quantitative understanding of cellular functions, biological questions are increasingly moving to the single molecule level and demanding techniques able to measure the dynamic behaviour of single biomolecules while undergoing conformational changes and binding events directly inside cells. Optical techniques for these single molecule studies typically use organic fluorophores as optical labels, which however suffer from bleaching and irreversible degradations. Metallic NPs have attracted increasing attention as alternative labels since they efficiently absorb and scatter light at specific wavelengths (called localised surface plasmon resonances - LSPR) and do not blink or photobleach. Importantly, the LSPR wavelength is very sensitive to the presence of another metallic NP in close proximity depending on the inter-particle distance. Metallic NPs attached to biomolecules can thus be used as "Plasmon rulers" of binding events with single molecule sensitivity by measuring the LSPR shift which occurs upon binding and formation of a NP dimer. However, all experiments reported to date failed to demonstrate biosensing with small NP dimers inside cells, since they rely on optical methods which are not background-free. In our laboratory, we have recently developed a novel FWM technique capable of resolving single small (< 40nm) metallic NPs background-free even in a highly scattering and fluorescing environment and operating at very low powers, hence compatible with live cell imaging. In this project we will pioneer the use of this novel FWM detection with NP dimers for local biosensing with single molecule sensitivity directly inside cells.
The interrelated research strand will combine FWM with electron microscopy (EM). This will support the biosensing strand by resolving NP dimers and enabling accurate measurement of their inter-particle distance owing to the sub-nanometer spatial resolution of EM. Moreover, CLEM is a unique tool to understand intracellular processes. For instance, cells have to make new proteins and transport these to the correct places to function properly. Likewise cells have to interpret signals from the outside and route them correctly. What carriers are being used to convey these signals? Where and how do proteins aggregate or segregate? Although dynamics can be studied by light microscopy, its limited resolution (>100nm) requires EM for precise localisation, but EM alone gives only a static image. In CLEM cells are labelled with markers and intracellular events are first followed in living cells with light microscopy. When an interesting event is observed, cells are rapidly fixed and processed for EM. Current approaches use fluorophores for light microscopy conjugated to gold NPs for EM. However, fluorophores photobleach, and when conjugated to metallic NPs might undergo quenching. In addition, there is an unknown modification between the last image from the light microscope and the fixed cell studied in EM since the EM processing procedures usually destroy the fluorophore. We will overcome these limitations by using our novel FWM detection as light microscopy in the CLEM process. FWM directly visualises bare metallic NPs without fluorophores, and is highly photostable and background-free. The culmination of this project will be the combination of both research strands to enable biosensing CLEM.

Who will benefit from this research?

i) The academic community. This is a highly cross-disciplinary project. The demonstration of Four-Wave Mixing sensing with individual small metallic nanoparticles acting as 'Plasmon rulers' will be very relevant to physicists and material scientists interested in localised plasmonic nonlinearities. Nonlinearity is key to giving plasmonics functionality, but is investigated experimentally by only a few groups worldwide due to the technical challenges of measuring the nonlinear response of single small metallic NPs with high spatial and temporal resolution. Hence our proposed research is timely and will significantly strengthen the UK's competitiveness in plasmonics. Moreover, we expect bioscientists striving to decipher complicated intracellular functions to be very excited by the possibility offered by FWM biosensing to directly detect local biomolecular binding events inside cells with unprecedented sensitivity. We also expect that the combination of FWM with electron microscopy, as a new correlative light electron microscopy approach, will impact researchers involved in microscopy technology developments from an instrument engineering point of view and as end-users.
ii) The commercial sector. This research specifically addresses the area of sensor technologies, and the development of novel imaging techniques for a better understanding of intracellular transport processes which are relevant for e.g. drug delivery. The direct involvement of BBInternational as project partner clearly shows that the proposed research contributes to the development of key industries in the biotechnology sector. Furthermore, the combination of electron microscopy with light microscopy is receiving much attention not only within the scientific community but also from light microscope manufacturers (Carl Zeiss and Leica Microsystems) and electron microscope manufacturers (FEI company).
iii) The public sector, through the benefit for public health in the development of novel technologies for a quantitative understanding of cellular behavior as a key step toward better diagnostics and medicines.

How will they benefit from this research?

The potential for impacts arising from the proposed work are:

- Academic Impact, via the scientific advancements in the FWM biosensing and CLEM developments, and through the cross-disciplinary training of a highly skilled Research Associate appointed in this project.

- Economic and Societal Impact, via the enhancement of the research capacity and knowledge of the project partner company BBInternational, and through the commercial exploitation of a FWM imaging module for CLEM with the electron microscope manufacturer company FEI. Moreover this research has the potential to improve health and quality of life by providing new tools for the quantitative understanding at the molecular level of cellular functions which regulate diseases and/or drug delivery.