Nonlinear plasmonic biosensing and functional imaging

Lead Research Organisation: CARDIFF UNIVERSITY
Department Name: School of Biosciences

Abstract

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.

Planned Impact

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.

Publications

10 25 50

 
Title Contemporary Dance OPTO-NANO 
Description The professional dancer and choreographer Jack Philp created a dance piece inspired by the research in my laboratory, with funding from the Arts Council Wales. This resulted in a showcase performance at the National dance House in Cardiff in November 2019 and in a video posted in social media. 
Type Of Art Performance (Music, Dance, Drama, etc) 
Year Produced 2019 
Impact The work has attracted considerable interest in social media. Several members of the general public attended the performance showcase which was followed by a question and answer session in which I had the opportunity to explain my research to the wider public. With the coreographer we plan to bring this forward into a dance tour and we are seeking further funding. 
URL http://www.jackphilpdance.co.uk/opto-nano
 
Description For an in depth quantitative understanding of cellular functions, biological questions are increasingly demanding techniques able to measure the behaviour of single biomolecules while undergoing binding events and/or conformationial changes directly inside cells. Optical techniques for these single molecule studies typically use organic fluorophores as optical labels, which however suffer from bleaching and irreversible degradation.
Metallic nanoparticles (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 interparticle 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.

In our laboratory, we have invented and developed an optical microscopy technique based on resonant four-wave mixing (FWM) capable of resolving single small 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 the research funded by this grant we have shown the use of this novel FWM detection with NP dimers, and demonstrated its potential for biosensing using dimers at variable interparticle distance as plasmon rulers.

Furthermore, we have correlated FWM with electron microscopy (EM) on a series of dimers. This not only demonstrated a novel correlative light EM technique, but also enabled accurate calibration of the dimer interparticle distance owing to the sub-nanometer spatial resolution of EM.

An additional outcome stimulated by this research was a key improvement on a method recently developed by us to quantitatively measure the absorption and scattering cross-section of single nanoparticles and determine particle asymmetry. This method has a strong commercial potential for particle manufacturers, and has strengthen our collaboration with the project partner from industry in this grant: BBI Solutions.
Exploitation Route These finding might be taken forward in several ways.

1) We intend to push forward the commercial impact of our method to quantify the absorption and scattering cross section of nanoparticles, applicable to any shape and material. We have submitted a patent application for IP protection, recently published, and we are working toward a knowledge transfer partnership with analytical instrumentation companies.
2) We intend to pursue the demonstration of correlative FWM EM microscopy as a novel research tool in the life science to understand intracellular processes. These can have non-academic impact in the pharmaceutical sector where NPs are widely used as drug-delivery vehicles.
3) We intent to push forward with future grant funding the detection of single small metallic nanoparticles background-free inside cells, using FWM, in particular via novel methodologies for single particle tracking.
Sectors Healthcare

Manufacturing

including Industrial Biotechology

Pharmaceuticals and Medical Biotechnology

 
Description The technology developed in this project has the potential of commercial impact via two routes 1) development of a new microscopy instrumentation for nanoparticle characterization, and 2) development of a new microscopy instrumentation for correlative light electron microscopy, with impact in the particle manufacturing as well as the pharmaceutical sector. We are having discussions with particle manufacturers, microscopy companies and manufacturers of analytical instrumentation, and plans for knowledge transfer are in place. Building from the results of this project, we have recently been awarded a major EU network bringing together 7 world-leading academic groups and 5 high tech companies at the forefront of optical microscopy and ultrafast laser technology developments merged with biomedical and pharmaceutical real-world applications, from 9 European countries. The network appoints 15 early stage researchers, trained to become the next-generation scientists in academia and industry, fostering global scientific and economic performance. We have also started an outreach project with the arts that resulted in a contemporary dance choreography inspired by this work being created and showcased at the National Dance Company House in Cardiff in 2019, with support from the Arts Council Wales.
First Year Of Impact 2013
Sector Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural

Economic

 
Description Experimental Equipment Call
Amount £731,951 (GBP)
Funding ID EP/M028313/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 03/2015 
End 03/2016
 
Description Initial Training Networks Call identifier: FP7-PEOPLE-2013-ITN
Amount € 584,513 (EUR)
Funding ID 607842 
Organisation European Commission 
Sector Public
Country European Union (EU)
Start 09/2013 
End 09/2017
 
Description Multiphoton Microscopy and Ultrafast Spectroscopy: Imaging meets Quantum (MUSIQ)
Amount € 4,034,447 (EUR)
Funding ID 812922 
Organisation Marie Sklodowska-Curie Actions 
Sector Charity/Non Profit
Country Global
Start 03/2019 
End 03/2023
 
Description TTL Integrated Biological Imaging Network Pump-priming Project: A novel correlative light electron microscopy (CLEM) technique to unravel the nano-toxicology of single gold nanoparticles in terrestrial isopods
Amount £24,808 (GBP)
Organisation United Kingdom Research and Innovation 
Department Technology Touching Life
Sector Public
Country United Kingdom
Start 09/2019 
End 03/2020
 
Description Tracking the motion of single nanoparticles inside living cells: New insights into intracellular crowdedness
Amount £80,000 (GBP)
Funding ID EPSRC DTP account 
Organisation Cardiff University 
Sector Academic/University
Country United Kingdom
Start 01/2021 
End 06/2024
 
Description Training Grant, Industrial CASE with BBI
Amount £94,126 (GBP)
Funding ID BB/L015889/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 09/2014 
End 09/2018
 
Description Welsh Government Award - LIFE SCIENCES BRIDGING FUND Optical Nanosizer: Wide-field extinction micro-spectroscopy for nanoparticle analysis
Amount £47,894 (GBP)
Funding ID LSBF/R6-005 
Organisation Government of Wales 
Sector Public
Country United Kingdom
Start 05/2017 
End 03/2018
 
Description BBI-nanoparticle analysis 
Organisation BBI Solutions
Country United Kingdom 
Sector Private 
PI Contribution We have developed a novel method to quantify the absorption and scattering cross section of single metallic nanoparticles using wide-field microscopy. The method enables simultaneous acquisition of hundreds of nanoparticles for statistical analysis. The method is simple, rapid, and quantitative. Precise characterisation of nanoparticle sizes and shapes is key to manufacturing quality control. Current techniques for particle characterisation are dynamics light scattering (DLS), which is easy to use but not very accurate and provides only the effective mean size (hydrodynamic radius), and transmission electron microscopy (TEM) which is time consuming and expensive. The optical extinction microscopy method developed by us overcomes these limitations and effectively complements these analysis techniques. BBI Solutions which is a world leading manufacturer of gold nanoparticles for lateral flow analysis and in-vitro diagnostics (http://www.bbisolutions.com/). They have a major nanoparticle synthesis and analysis headquarter at Cardiff. They see huge potential benefit in the use of optical extinction for particle analysis and quality control, owing to the accuracy and versatility of the method which can be implemented cost-effectively on a conventional wide-field microscope and is applicable to any nanoparticle.
Collaborator Contribution BBI Solutions has provided us with nanoparticle materials, including custom made, and access to their analysis tools. They have dedicated a significant amount of time in technical discussions with us. They have been industrial partners on an EPSRC funded project, and are co-investigators in a BBSRC iCASE studentship with us where they have hosted the PhD student on-site for a significant amount of time.
Impact This collaboration was aimed at providing BBI solutions with a cost-effective tool for particle analysis which they can use instead of costly TEM, hence having a clear financial benefit. We also explored with them ways in which they could expand their portfolio of nanoparticle manufacturing, using our quantitative analysis to provide better product specification to customers and improve nanoparticle design.
Start Year 2013
 
Title ANALYSING NANO-OBJECTS 
Description Methods and apparatus for analysis of nano-objects using wide-field bright field transmission techniques are described. Such methods may comprise acquiring a plurality of images of a sample (124) comprising a plurality of nano-objects using bright field illumination via a continuously variable spectral filter (114), and identifying a nano-object within the sample in the plurality of images, wherein the position of the nano-object changes between images. Using data extracted from the plurality of images, an extinction cross-section of the identified nano-object may be quantitatively determined. 
IP Reference WO2019020975 
Protection Patent application published
Year Protection Granted 2019
Licensed No
Impact This development has resulted in a number of research papers that have been published in high quality international journals. We are seeking to engage with companies manufacturing analytical instrumentation to licence the IP.
 
Title Quantitative Extinction analysis 
Description We have developed an optical extinction microscopy analysis method to quantify the optical extinction cross-section s_ext of single nanoparticles using wide-field microscopy. The method utilities the simultaneous acquisition of hundreds of nanoparticles for statistical analysis. The method is simple, rapid, and quantitative. We have demonstrated a sensitivity of s_ext below 1nm^2 corresponding to the detection of a single gold nanoparticle of 2nm diameter, using only ~1min total acquisition time. From the measurement of s_ext and its dependence on the light polarization, an accurate quantification of particle sizes and asymmetries at the single particle level is rapidly obtained for a statistically-relevant number (>100) of particles. An Extinction Suite Macro for ImageJ was also developed, for quantitative data analysis. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2013 
Impact This technique has generated commercial interested with our industrial collaborators BBI Solution, a world leading nanoparticle manufacturer. Precise characterisation of nanoparticle sizes and shapes is key to manufacturing quality control. Current techniques for particle characterisation are dynamics light scattering (DLS), which is easy to use but not very accurate and provides only the effective mean size (hydrodynamic radius), and transmission electron microscopy (TEM) which is time consuming and expensive. The optical extinction microscopy method developed by us overcomes these limitations and effectively complements these analysis techniques. Malvern Panalytical Ltd manufacturing analytical instrumentation including DLS, is interested in a knowledge transfer partnership on this technology. 
URL http://langsrv.astro.cf.ac.uk/Crosssection/Extinction_Suite/Extinction_Suite.html
 
Title Resonant FWM imaging 
Description We have developed a novel optical microscopy technique to visualise small single metallic nanoparticels (NPs), without the need of any fluorophore labelling, in a very specific and background free way, even in highly scattering environments such as cells and tissues. In this method individual small (radii below 20nm) metallic NPs are visualised with high contrast and photo-stability, free from background and at powers corresponding to negligible photothermal heating, via their coherent nonlinear optical emission called Four-Wave Mixing (FWM) in resonance with the NP surface plasmon. The technique has been implemented to work both in transmission and reflection geometry. It includes a polarisation resolved analysis to detect particle asymmetry, a phase resolved analysis for biosensing, and a time-resolved analysis to quantify thermal and mechanical couplings of NPs with their environments. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2012 
Impact The technique is being utilized extensively over several research projects, including a collaborative project with Bristol University with interest in correlative light-electron microscopy . It is planned to become part of an imaging facility at Cardiff University. 
URL http://www.cardiff.ac.uk/people/view/81122-borri-paola
 
Title Single particle tracking via four-wave-mixing optical vortex interferometry 
Description We have developed and demonstrated a new four-wave-mixing interferometry technique, whereby the position of a single nonfluorescing gold nanoparticle is determined with nanometric precision in 3D from rapid single-point measurements at 1-ms acquisition time by exploiting optical vortices. The technique is also uniquely sensitive to particle asymmetries of only 0.5% ellipticity, corresponding to a single atomic layer of gold, as well as particle orientation. This method opens new ways of unraveling single-particle trafficking within complex 3D architectures. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2015 
Impact Ultimately, this new method paves the way towards a new form of single-particle tracking, where not only the nanoparticle position but also its asymmetry, orientation, and chirality are detected with submillisecond time resolution, revealing much more information about the nanoparticle and its complex dynamics (e.g., hindered rotation) while moving and interacting within a disordered 3D environment. As such it can have many applications in physical science systems (e.g. diffusion in complex liquids and gels, liquid crystals) and in the life sciences (single-molecule intra-cellular trafficking). 
 
Description Cardiff Science Festival 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact A stand showcasing our group research activity to the general public was set-up at the Cardiff Science Festival on 19 -20 July 2014. At this stand four members of our group (two postdoctoral researchers and two PhD students) gave practical demonstrations and gifts, and presented an "attention grabbing" poster based around the concept of "Blinged Nanoparticles"
Year(s) Of Engagement Activity 2014
URL https://www.facebook.com/Cardiff-Science-Festival-386755174684198/
 
Description Contemporary Dance project OPTO NANO 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact In collaboration with a contemporary dance professional choreographer Jack Philp, an outreach project was started aimed at creating a dance piece inspired by the biophotonics research in my lab. Jack was awarded a grant from the Arts Council Wales to work on the creation of the piece which was showcased at an open rehearsal to the public at the National Dance House in Cardiff in November 2019. The work is titled OPTO-NANO and resulted in a promotional video and posting on social media. Jack is working toward expanding the work into a tour and we are seeking further funding.
Year(s) Of Engagement Activity 2019
URL http://www.jackphilpdance.co.uk/opto-nano