Multiphoton microscopy of lipid-protein dynamics in living cells using correlative Coherent Antistokes Raman Scattering and Two-Photon Fluorescence

Lead Research Organisation: Cardiff University
Department Name: School of Biosciences

Abstract

The purpose of this research is to develop a new generation optical microscope able to resolve the dynamics of formation, trafficking and breakdown of lipid droplets (LD) and associated proteins in living cells. LD development plays a major role in obesity and diabetes which pose a substantial burden on modern developed countries health budgets. Fluorescence microscopy, using antibodies labelled with dyes or fusion of proteins with fluorescent tags has provided a highly sensitive and specific method of visualizing proteins in cells. Fluorescent markers for optical microscopy of lipids are available, however suffer from rapid photobleaching, that is an irreversible degradation of the fluorescence intensity after excitation with light. Moreover, the cellular modifications arising from the addition of fluorescent lipid probes and lipid staining processes raise major questions if the observed behaviour is real or artefactual. As a result the dynamics of LD development and breakdown has been very difficult to study with fluorescence microscopy, and accurate quantification often impossible. The key idea of the microscope being developed in this project is to obtain the image contrast from lipids via the scattering of light by the vibrations in the lipid chemical bonds (Raman scattering). In this way, the lipids present in cells are visualised without the need of labelling. Raman scattering can be enhanced when using two short laser pulses to excite the vibrations and generate Coherent Antistokes Raman Scattering (CARS). CARS depends nonlinearly on the exciting light intensity, so that sufficient intensities for CARS generation are achieved only in the small focal volume where the exciting photons are concentrated. This results in a very high three-dimensional spatial resolution and lipid droplets of submicron size can be examined with this method. The microscope will be designed to observe lipids on the inside of LDs with CARS, and simultaneously visualise the fluorescence from tagged-proteins associated to the outside of the LD, so that the complete picture of LD development can be determined in living cells. We have identified a number of candidate proteins that have an effect on LDs. This microscope will allow us to investigate the mechanisms behind the interactions of these proteins and LDs in a way that was previously not possible. The identification of the proteins responsible for LD homeostasis in human cells will play a key role in designing therapeutic strategies to control the cellular lipid content. Thus a longer term goal of the project is the development of a high-content screening version of the microscope to discover new targets involved in LDs. Besides the researchers directly involved in this project, several other scientists at Cardiff University are interested in cellular lipids in relation to e.g. atherosclerosis, pulmonary cells producing surfactants preventing lung collapse, breast development and associated milk production. These researchers would greatly benefit from the availability in-house of a CARS/fluorescence microscope dedicated to lipid biology, including a high-content screening version. Results of this work will be published in international journals, so that researchers from both physics and biological disciplines worldwide will benefit from these outcomes. The usage of this microscopy technique will be of relevance in medical applications, for drug discovery and to improve the diagnostic and treatment of lipid-related health problems. Additionally, the proposed research contains the realization of an economic design of the CARS microscope for its possible widespread application. With no CARS microscope commercially available, we expect microscope manufacturers to be interested in its exploitation. Also laser manufacturing companies will be interested in the realisation of laser sources optimised for CARS/multiphoton microscopy. In fact one such company is a collaborator on this project.

Technical Summary

Our purpose is to develop correlative Coherent Antistokes Raman Scattering (CARS)/Two Photon Fluorescence (TPF) microscopy to resolve the dynamics of formation, trafficking and breakdown of lipid droplets (LD) and associated proteins in living cells. LD development plays a major role in obesity and diabetes which pose a substantial burden on modern countries health budgets. Fluorescent markers for optical microscopy of LDs are available, yet have a tendency to photobleach rapidly even under moderate illumination. As a result the spatio-temporal dynamics of LD development and breakdown have yet to be determined. In CARS microscopy, the image contrast from lipids is obtained via the scattering of light by the vibrations in the lipid chemical bonds. In this way, lipids intrinsically present in cells are visualised without the need of labelling. CARS is a multiphoton process with very high three-dimensional spatial resolution so that lipid droplets of submicron sizes can be examined. Although CARS microscopy applied to cell biology has been reported in the recent literature, there is no commercially available CARS microscope to date. In this project, we will develop a new generation CARS/TPF microscope based on a single laser, for an economic design, and utilising a differential excitation/detection method to improve CARS image contrast. The microscope will be designed to observe lipids on the inside of LDs with CARS, and simultaneously visualise the fluorescence from tagged-proteins associated to the outside of the LD, for a complete picture of LD development in living cells. We have identified a number of candidate proteins (of the COPI and COPII families) that have an effect on LDs. This microscope will allow us to investigate the mechanisms behind the interactions of these proteins and LDs in a way that was previously not possible. A longer term goal of the project is the development of a high-content screening to discover new targets involved in LDs.

Planned Impact

The current first world 'obesity epidemic' is a result of a reduction in physical activity and increase in calorie consumption as changes in modern lifestyle take place. Lipid storage mechanisms contribute to obesity. This project addresses an important shortfall in current methodologies for accurate analysis of the life cycle of a lipid droplet, and the timescale of associated protein interactions. It will have a major impact on basic research in lipid droplet dynamics (within 3-5years) and is likely to feature in clinical diagnosis of lipid storage related genes (5-10yrs). By identifying new mechanisms involved in cellular lipid storage with CARS microscopy, we will discover new targets for the design of therapeutic entities that will enable us to gain control of the cell's lipid storage cycle and improve lipid-related health problems. With no CARS microscope commercially available to date, we expect microscope manufacturers to be interested in the exploitation of the economic CARS microscope design (based on a single laser) developed in this project. Also laser manufacturing companies will be interested in the realisation of sources optimised for CARS/multiphoton microscopy, and one such company is a collaborator in this project. We anticipate markets for the high-content screening CARS technology in commercial drug discovery and therapeutic development, as well as a potential for diagnosis in both public and private sectors (5-10yrs). The contribution of this project to these beneficiaries and to the nation's health, wealth and culture will be mainly through: 1) Knowledge: via the scientific advancements in the understanding of intracellular lipid metabolism and the CARS/multiphoton technology development. 2) People: The RA on this project will receive sate of the art training in photonics. 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. All applicants teach and/or organize modules on this course. The microscopy technology developed in this project will add to the depth of the research training offered by this course. This will increase the impact and reputation of the course and will allow better recruiting of excellent students to be trained as Biophotonics leaders of tomorrow. 3) Improvement of Health and quality of life. The direct research output will lead to greater understanding of lipid metabolism and reveal new therapeutic targets. We will focus our high content siRNA screen on protein kinases and phosphatases as they make very 'druggable' targets and are currently subject to intensive drug development by companies. This is likely to provide a big reduction in lead time for translation of our research outcome into therapeutic strategies. This gain in cost-effectiveness will ultimately impact on time and development costs, providing a substantial benefit for public health in the prevention and/or cure of lipid-related health problems. Cardiff School of Biosciences has appointed a Head of Innovation, Partnership and Engagement to maximize coordination of commercialization activities. Based on its track record the School is one of three Knowledge Transfer Partnerships (KTP) champions. With direct help from the University KTP team it will run a series of showcases to attract partnerships with companies. Biophotonics is an area of strength of the School, and the outcomes of this research project will be promoted at these events. The School also organises an 'Imaging Symposium' focused on microscopy as a regular annual event which will provide the ideal opportunity to communicate the outcomes of this project to industrial beneficiaries. Links with industrial partners will also occur through MSc projects hosted by companies, and through the initiation of CASE PhD studentships. The School also supports a range of public engagement activities.

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 Multi-photon microscopy based on Coherent Antistokes Raman Scattering (CARS) has emerged in the last decade as a powerful novel technique offering non-invasive, rapid microscopic examination of cells and subcellular structures under physiological conditions with high chemical contrast without the need of labeling. The main achievements of this grant are:

i) The design and construction of a second-generation CARS microscope (including in-house developed software for data acquisition and analysis) featuring enhanced chemical contrast via dual-frequency/differential CARS and hyperspectral CARS, multimodal CARS and fluorescence simultaneous acquisition, and a compact user-friendly design with a single broadband laser.
ii) High-resolution three-dimensional imaging of COPII coat-protein components co-localised with lipid droplets in living mammalian cells using correlative fluorescence of labelled proteins and CARS of label-free lipid droplets. This has progressed into a subsequent analysis, through hyperspectral CARS, of changes in lipid-droplet composition in the presence or absence of this protein coat.
iii) Quantitative determination of the chemical composition of lipid droplets label-free in live adipose-derived human stem cells differentiating into adipocytes using differential CARS and hyperspectral CARS.
iv) The demonstration of a high-content high-throughput CARS imaging platform, using Bessel beam illumination and sparse sampling acquisition.
v) The application of hyperspectral CARS to imaging 3D multicellular structures (organoids).
Exploitation Route CARS microscopy is a powerful technique relevant to a number of biomedical applications. Its ability to visualize lipids label-free inside living cells has the potential to become a unique tool for the diagnosis and treatment of lipid-related diseases including atherosclerosis, diabetes and obesity. Our recent finding about the relevance of lipid droplets in mouse oocytes for their development into good quality embryos could pave the way to use CARS as a powerful label-free tool in IVF clinics. We have also started a new collaboration using CARS to investigate the accumulation of lipids in brain tumour (glioblastoma) with the potential to be taken forward in biomedical research.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description The technology developed in this project has been applied to many biological questions, with the potential to advance biomedical research and the healthcare sector. For example, work started with this project has continued and was taken forward to characterize lipid droplets in mouse oocytes and early embryos. We are exploring whether the spatial distribution and composition of lipid droplets can be used as markers of egg quality and embryo developmental potential for possible use in IVF clinics. The development of an advanced quantitative data analysis software associated with the microscopy technique in this project (hyperspectral CARS) is also attracting much attention from pharmaceutical companies and the biotech sector. We expect this software to become a powerful tool of widespread use for quantitative chemical imaging.
First Year Of Impact 2011
Sector Healthcare,Pharmaceuticals and Medical Biotechnology
Impact Types Cultural,Societal

 
Description A novel imaging approach to track brain cancer metabolism - ISSF3 Collaboration Panel: Cross-Disciplinary Award, Cardiff University
Amount £22,545 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 09/2019 
End 04/2020
 
Description Addressing the dietary needs of Kazakhstan: Developing low-cost omega-3 fatty acid production by microalgae using CARS microscopy
Amount £91,269 (GBP)
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2016 
End 12/2018
 
Description Creating super-scattering Raman-active genetically encoded proteins
Amount £198,490 (GBP)
Funding ID EP/V048147/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 01/2021 
End 06/2022
 
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 04/2015 
End 03/2016
 
Description ISSF3 Cross-Disciplinary Award: Uncovering metabolic errors in foetal brain development through advanced lipid imaging
Amount £37,067 (GBP)
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 11/2020 
End 05/2021
 
Description Lipid droplets in oocytes: shedding new light on why fats are good or bad for development.
Amount £472,355 (GBP)
Funding ID BB/P007511/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2020
 
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 04/2019 
End 03/2023
 
Description Non-invasive chemically-specific imaging of lung inflammation
Amount £24,984 (GBP)
Funding ID UKRI Technology Touching Life IBIN 
Organisation King's College London 
Sector Academic/University
Country United Kingdom
Start 05/2021 
End 10/2021
 
Description Royal Society Challenge Grant Awards
Amount £91,269 (GBP)
Funding ID CH160031 
Organisation The Royal Society 
Sector Charity/Non Profit
Country United Kingdom
Start 12/2016 
End 12/2017
 
Description Science Committee - Multidisciplinary Project Award
Amount £403,597 (GBP)
Funding ID C368/A22099 
Organisation Cancer Research UK 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2016 
End 07/2019
 
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 GSK
Amount £95,042 (GBP)
Funding ID BB/N50371X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 10/2015 
End 09/2019
 
Description CRS on organoids with Cellesce 
Organisation Cellesce Ltd
Country United Kingdom 
Sector Private 
PI Contribution Expertise in optical microscopy technology developments beyond state of the art, in particular label-free microscopy based on Coherent Raman Scattering, and associated quantitative image analysis. Application of CRS microscopy to organoid samples provided by the company.
Collaborator Contribution Cellesce has a patented technology for the expansion of human-derived "organoids" at scale for high-throughput screening in the pharmaceutical industry. Organoids are 3D clusters of cells, derived from patient biopsies, typically from tumours, that accurately represent the architecture and physiology of the original tissue. When grown on a commercial scale, organoids represent a new, improved and transformational, model for drug discovery and for clinical and academic research. In this partnership, Cellesce is providing us with living and fixed organoids samples, to be investigated with our optical microscopy technologies beyond state of the art.
Impact Presentations at conferences and meetings. New grant applications. The collaboration is multi-disciplinary at the physics-life sciences interface.
Start Year 2019
 
Title Differential CARS microscopy 
Description Dual-beam/differential CARS microscopy is an multiphoton microscopy technology that enables high speed label-free imaging of biomolecules using Coherent Antistokes Raman Scattering (CARS) by driving and detecting two vibrational resonances simultaneously, hence with improved chemical specificity and contrast compared to more conventional single frequency CARS systems. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2009 
Impact Improved chemically specific imaging of lipids in living cells 
 
Title Hyper spectral image analysis software 
Description The software developed is an image analysis tool to quantitatively retrieve concentrations of chemical components and spectral Raman-like profiles from hyperspectral CARS microscopy data 
Type Of Technology Software 
Year Produced 2013 
Impact Quantitative determination of concentration of chemical components (lipids, proteins, DNA) and their spatial distribution in cells and tissues from label-free hyperspectral CARS microscopy data. The software has been made freely available, and has been utilized by several groups. It is also applicable beyond CARS data, to any dataset that requires a decomposition analysis (eg fluorescence spectra). It is becoming part of an image analysis suite embedded in an institutional image facility. 
URL http://langsrv.astro.cf.ac.uk/HIA/HIA.html
 
Title single-source hyperspectral CARS microscopy 
Description The technology developed is an improved CARS microscopy set-up enabling to probe a large range of vibrational resonances using a single sub-10fs broad-band Ti:Sa laser source, for compact, cost-effective and more user-friendly CARS microscopy applications 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2011 
Impact Improved label-free chemically-specific imaging of lipids, proteins and DNA in cells and tissues. The technique is now being utilized by other groups in our department. It is planned to become part of an institutional imaging facility over the coming year. 
URL http://www.cardiff.ac.uk/people/view/81122-borri-paola
 
Description BioNanoPhotonics Symposium 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact This is a scientific symposium that I organise every two years, with international invited speakers and participation from industry and postgraduate students. We started having 40 delegates in 2011 and reached 80 delegates in 2015. The event has sparked many scientific discussions and further collaboration with industry among the delegates.
Year(s) Of Engagement Activity 2011,2013,2015,2017
URL http://www.bionanophoto.org.uk/
 
Description CARS Imaging for high-thoughput microscopy 
Form Of Engagement Activity A press release, press conference or response to a media enquiry/interview
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Automated high throughput microscopy is an invaluable tool for assessing large numbers of biological samples. In this article to the Biotech International magazine we discuss the potential of Coherent Antistokes Raman Scattering (CARS) microscopy as a chemically sensitive label-free imaging technique for high throughput in order to obtain chemical informations from a large number of biological samples. Article printed in the April/May 2011 volume 23 of the Biotech International magazine for the life science industry

no actual impacts realised to date
Year(s) Of Engagement Activity 2011
 
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