Autofluorescence lifetime metrology for label-free readouts of heart disease and arthritis

Lead Research Organisation: Imperial College London
Department Name: Dept of Physics

Abstract

This project aims to provide new label-free detection and imaging tools for minimally invasive diagnosis of arthritis and heart disease. When biological tissue is illuminated with light at appropriate wavelengths, certain naturally occurring biomolecules can absorb this excitation energy and emit new radiation called fluorescence. By analysing such autofluorescence signals, it is possible to detect particular biochemical or structural changes in tissue, which may be exploited to detect the early onset of diseases such as arthritis, heart disease and cancer, which can cause changes in the concentration, distribution and interaction of these autofluorescent biomolecules. Unfortunately, biological tissue is heterogeneous, typically containing many kinds of biomolecule in unknown quantities that can interact with autofluorescence measurements. It also strongly scatters optical radiation, making quantitative fluorescence measurements unreliable. It is therefore desirable to analyse tissue autofluorescence in a way that avoids artefacts arising from unknown variations in molecular concentrations and light intensity. One way to do this is to exploit the fact that excited molecules can radiate fluorescence at different rates, depending on the particular biomolecule or on how it is interacting with its surroundings. By observing the fluorescence decay times (lifetimes), it is possible to distinguish different chemical species or different molecular environments that can correlate with different tissue structures. Although this technique is attracting increasing interest in laboratory-based research, there is very little work, to date, on translating this to clinical practice. Here, we propose to develop fibre-optic-based probes that clinicians can use to measure fluorescence lifetimes in situ in biological tissue samples or live subjects including patients. We will also develop a fluorescence lifetime imaging (FLIM) system that will be able to rapidly map the spatial variation of autofluorescence lifetime, which can provide diagnostic functional images for medical research and clinical practice.We will develop one fibre-optic autofluorescence lifetime (AFL) probe to be applied to changes in tissue matrix components associated with arthritis, which leads to loss of joint function and pain in ~9 million people in the UK's aging population. Arthritis is caused by the degradation of cartilage in our limb joints. Currently, there is no way to examine the integrity of cartilage tissue without invasive surgical biopsy. In preliminary work, we have shown that degraded cartilage exhibits significantly different AFL compared to healthy cartilage. Here we aim to investigate how AFL measurements can provide label free information on the structure and health of cartilage. We would also investigate the prognostic value of FLIM arthroscopy of cartilage in early rheumatoid and osteoarthritis and monitor the effect of drugs applied to repair cartilage.A second AFL probe will be applied to the study of heart disease - the major cause of death in the developed world - which is characterised by abnormalities of heart muscle energetics. Energetic inefficiency accelerates further disease progression, manifesting as changes in the mechanical and electrical properties of the heart. Two important autofluorescent molecules, NAD(P)H and flavins, are intimately involved in metabolism but, apart from our preliminary results presented below, no AFL measurements have been made in situ in live heart tissue. We aim to investigate how cardiac AFL can provide a quantitative label-free readout of the metabolic state of the beating heart and to identify signatures of disease through AFL measurements and FLIM of cardiac tissue matrix adaptations associated with heart disease and electrical disturbances. These label-free readouts of diseased heart tissue could provide a novel means to determine treatment of patients following a heart attack.

Planned Impact

This project aims to provide new non-invasive tools for improved patient prognosis and treatment, particularly for arthritis and heart disease, and so addresses the needs of the aging UK population. The autofluorescence lifetime (AFL) technologies to be developed and validated would find other biomedical applications, e.g. for research, diagnosis and monitoring of therapies in cancer, inflammation, wound healing and any condition where label-free readouts of tissue matrix properties or metabolism are useful. They could also be used with fluorescence labels, including in genetically manipulated live disease models to provide readouts of signal pathways for drug discovery. Other bio-applications include tissue engineering, stem cell culture and bio-reactors. Some of these are being addressed by lab-based FLIM microscopy but our fit-for-purpose technology should lower the cost and increase the potential for real-world deployment. AFL technologies are applicable in the pharmaceutical sector, e.g. to test drugs in patients and animals - permitting longitudinal studies and therefore reducing the required numbers of animals. For agriculture, AFL may be useful to study plant disease, to monitor the effects of chemical intervention and to assess crops before harvest. For food safety, AFL may help detect dangerous bacteria or decomposition. Beyond the life sciences, our proposed instrumentation could be used to rapidly assess, in situ, devices such as OLED's and photovoltaics, including during manufacturing for quality control. The most direct impact of this project is envisaged to be medical research addressing the label-free detection, diagnosis and monitoring of arthritis and heart disease, although we anticipate patient-orientated follow-on studies and clinical trials to deliver new clinical tools. We also note the valuable training of project research staff. For both arthritis and heart disease, there are unmet needs for minimally invasive functional and spatially-resolved readouts of biological tissue to improve patient prognosis and more effectively target treatment. For arthritis, the proposed AFL instrumentation could enable the early detection of disease - before significant irreversible cartilage degradation. This would help the NHS budget by reducing the number of joint replacements and significantly improve patient quality of life. The ability to monitor cartilage integrity in situ also enables monitoring of potential therapies such as inhibitors that block the action of cartilage-degrading enzymes that otherwise can only be studied using invasive biopsy and/or the sacrifice of animals at each endpoint. Currently there are no such therapies targeting cartilage degradation available for arthritis. For heart disease the use of AFL to read out changes in metabolism and tissue matrix properties could address the current inability to accurately predict which heart disease patients are at risk of both sudden cardiac death from malignant arrhythmias and progressive mechanical deterioration and death from 'pump failure'. Better patient selection and targeting of expensive treatments, such as implantable defibrillators, arrhythmia ablation and the emerging stem cell and gene therapies, would significantly improve patient outcomes and reduce costs to the NHS. Because this AFL instrumentation represents a relatively cost-effective add-on to current clinical diagnostic and therapeutic approaches, it presents new commercial opportunities, e.g. for medical device manufacturers. Kentech Instruments Ltd, in particular, are keen to develop products for clinical AFL imaging based on their gated intensifier technology and will work with us to establish the requirements for clinical devices. For the single-point AFL fibre-optic probe, we are seeking commercial exploitation partners and want to use the data from this project to validate the clinical applications and strengthen our case for commercial investment.

Publications

10 25 50
 
Description During this multidisciplinary research project, we developed two portable fibre-optic probe-based instruments for in situ single point measurements of biological tissue utilising readouts based on autofluorescence signatures and reflected white light. Autofluorescence is the emission from naturally occurring fluorophores in biological tissue that can excited with ultraviolet or visible radiation. These instruments can resolve tissue autofluorescence with respect to wavelength and can also measure the decay time of this emission, which typically occurs on a nanosecond timescale. This combined autofluorescence spectral and fluorescence lifetime information can provide information about changes in tissue matrix properties, such as scarring (which is manifest by an increase in collagen) or degradation of collagen such as occurs during osteoarthritis. These instruments can also read out changes in cellular metabolism, which can be used to study the function of the heart or can be used to identify cancerous tissue.

The technology used to make these measurements is based on spectrally resolved time-correlated single photon counting (TCSPC) following excitation with gain-switched picosecond diode lasers. Essentially these instruments deliver ultraviolet or blue ultrashort optical pulses via an optical fibre that excites autofluorescence which is collected by other optical fibres incorporated into the same optical probe. This emission is spectrally filtered and sent to time-resolved detectors to allow us to measure the decay profiles of the autofluorescence signals resulting from the pulsed excitation. The system we have developed consists of compact instrumentation that fits on a small trolley and plugs into a laptop computer.

We have applied one of these instruments to study heart disease and particularly the changes in tissue autofluorescence following a heart attack. Through preclinical experiments using rodent models, we have established that our instrument can be used to detect scarring and changes in the energetics of the heart tissue. If this could be implemented with human patients, our results suggest that the clinicians could gain valuable information about how a specific heart recovers after a heart attack - which could inform the treatment regime of that particular patient. The system also has the potential to provide a readout of the metabolic health of a heart in real time during open heart surgery.

We have also applied one of these instruments to studies of the degradation of cartilage associated with osteoarthritis. First we showed that our instruments could provide quantitative readouts of controlled degradation of cartilage tissue in vitro and then we applied the instrument to ex vivo animal and human tissue and showed that we see a change in the autofluorescence lifetime of the cartilage when it degrades. This is important because clinicians currently lack a means to diagnose osteoarthritis before significant symptoms are presented, by which time it is usually too late for any effective treatment. Our instruments could potentially be used to help diagnose osteoarthritis earlier and to monitor the effectiveness of new treatments.

These optical fibre-based probes provide single point measurements and so do not yield information about tissue structure or sub-mm scale spatial variations in the tissue properties. For exploratory diagnosis and screening applications, we therefore developed a wide-field fluorescence lifetime imaging (FLIM) endoscope system that can acquire maps of autofluorescence lifetime cross a field of view of a few mm. We showed that this was able to differentiate between normal tissue and cancer in ex vivo tissue. Such experiments usually require bulky apparatus but we developed a compact trolley-based FLIM endoscope system that can be used for clinical or preclinical imaging with a handheld optical fibre-coupled imaging probe.

We also studied the autofluorescence lifetime signals in a novel automated FLIM microscope where we showed that the change in lifetime of cellular autofluorescence could be used to read out the response of the cells to anticancer drugs.
Exploitation Route Our single-point instruments and the wide-field FLIM endoscopes can be used to provide label-free optical molecular contrast to study a range of diseases including cancer, heart disease and osteoarthritis. Our results indicate that these instruments can provide signals that permit diseased tissue to be identified and this could be used for diagnosis and screening, e.g. using "optical biopsy", or to guide conventional biopsies. They could potentially be used to assay the response of specific patients to particular therapies and therefore to stratify patients for "personalised medicine".

The next step is to translate the preclinical and ex vivo results to clinical trials in humans. We already have the instrumentation in place and so the next studies must concentrate on obtaining the data required to obtain ethical and regulatory approval. The key issue is that there is no data on hazards or regulation of light dose to internal tissues. Our instruments typically present lower intensities and therefore lower hazards than sunlight and are well within the safe exposure limits for skin.
Sectors Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description We are working to develop new projects utilising the instruments developed during this project. In particular, we have established a collaboration with a cardiac surgeon at the NHLI and aim to take the fibre optic point probe instrument into the operating theatre to demonstrate its first-in-man use. The osteoarthritis work has now been published and we are seeking funding for a clinical study in patients.
First Year Of Impact 2015
Sector Healthcare
 
Description part of Marie (Sklodowska-)Curie actions research fellowships
Amount € 3,200,000 (EUR)
Organisation European Union 
Sector Public
Country European Union (EU)
Start 10/2014 
End 12/2017
 
Title Characterization Of Nad(P)H And Fad Autofluorescence Signatures In An Isolated-Perfused Rat Heart Model 
Description Raw data concerning publication titled "Characterization of NAD(P)H and FAD autofluorescence signatures in an isolated-perfused rat heart model" Abstract Autofluorescence spectroscopy is a promising label-free approach to characterize biological samples with demonstrated potential to report structural and biochemical alterations in tissues in a number of clinical applications. We report a characterization of the ex vivo autofluorescence fingerprint of cardiac tissue, exploiting a Langendorff-perfused isolated rat heart model to induce physiological insults to the heart, with a view to understanding how metabolic alterations affect the autofluorescence signals. Changes in the autofluorescence intensity and lifetime signatures associated with reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) and flavin adenine dinucleotide (FAD) were characterized during oxygen- or glucose-depletion protocols. Results suggest that both NAD(P)H and FAD autofluorescence intensity and lifetime parameters are sensitive to changes in the metabolic state of the heart owing to oxygen deprivation. We also observed changes in NAD(P)H fluorescence intensity and FAD lifetime parameter on reperfusion of oxygen, which might provide information on reperfusion injury, and permanent tissue damage or changes to the tissue during recovery from oxygen deprivation. We found that changes in the autofluorescence signature following glucose-depletion are, in general, less pronounced, and most clearly visible in NAD(P)H related parameters. Overall, the results reported in this investigation can serve as baseline for future investigations of cardiac tissue involving autofluorescence measurements. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
 
Description Kentech Instruments Ltd 
Organisation Kentech Instruments Ltd
Country United Kingdom 
Sector Private 
PI Contribution We have advised Kentech on improvements to be made to their gated optical image intensifiers and related components and have tested prototypes. We have demonstrated that their gated intensifiers can be integrated into a range of optical instruments, particularly for fluorescence lifetime imaging (FLIM) for biomedical applications, and this has led to increased sales for them. We collaborated with them on an EPSRC Healthcare Partnership project that built on earlier collaborations with Kentech and also on two MRC projects to develop FLIM instrumentation.
Collaborator Contribution Kentech Instruments Ltd advised us (with no charge) on the design of FLIM systems using gated optical image intensifiers and supplied us with a custom device for handheld FLIM that we designed in collaboration. They have sold us customised gated intensifier systems and refined the specifications of their products at our request.
Impact Joint publications Feedback to Kentech's product development
 
Title Lower cost fluorescence lifetime measurement system 
Description While the single-point autofluorescence endoscopic (SAFE) probe is compact and relatively low cost compared to a laser scanning endoscope, it is still relatively large and expensive for widespread clinical application. Since part of our exploitation strategy is to collect more clinical autofluorescence lifetime data in a number of different clinical contexts, we would like to replicate our SAFE functionality ion lower costs systems that could be deployed in parallel in different hospital settings. Accordingly, we have worked to reduce the cost of the electronic circuitry required to determine the autofluorescence lifetimes. For this we have developed a relatively simple frequency domain system utilising excitation light from a blue GaN diode laser and have implemented frequency domain detection using a programmable FPGA. This has the potential to significantly reduce the cost and the size of our instrumentation such that we can make a number of low-cost systems for parallel clinical trials. This more compact and cheaper instrumentation would also have better prospects to be translated to clinical practice. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2014 
Impact We developed a relatively simple fluorescence lifetime measurement system utilising excitation light from a blue GaN diode laser and have implemented low cost detection using a programmable FPGA. This has the potential to significantly reduce the cost and the size of our instrumentation such that we can make a number of low-cost systems for parallel clinical trials. This more compact and cheaper instrumentation would also have better prospects to be translated to clinical practice. 
 
Title Single-point autofluorescence endoscopic (SAFE) probe 
Description We developed a compact single-point autofluorescence endoscopic (SAFE) probe to measure the spectral and lifetime properties of tissue autofluorescence in vivo. This instrument can enable spectrally resolved lifetime measurements via a specially designed fibre-optic probe that is 2.4 mm diameter such that it can pass through the (2.8 mm) biopsy channel of a standard clinical endoscope. It has been engineered to be self-contained on a trolley and is implemented with gain-switched picosecond diode lasers for excitation in the u.v. and blue. FLIM is implemented with time-correlated single photon counting (TCSPC) and spectral discrimination is provided by either a spectrometer or dichroic beam splitters and filters. We have also incorporated a spectrometer to record the diffuse reflectivity spectrum for white light illumination. This multimodal single-point fibre-optic probe thus provides real-time measurements of tissue properties and, because it bins all the photons detected from a ~mm area of tissue into only a few detection channels, provides high signal to noise ratio with sufficient numbers of detected photons to permit (spectrally-resovled) analysis of complex fluorescence decay profiles, such as are typically encountered with tissue autofluorescence. This instrument has been successfully applied in clinical trials of skin cancer (at Lund University Hospital) and of GI cancer (at Charing Cross Hospital). It is straightforward to use during clinical procedures and will continue to be used in clinical investigations. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2012 
Impact The successful trial with this instrument led to further trials with similar instruments being undertaken funded by a new EPSRC project. 
 
Title Time-resolved fluorometer to read out changes in plant metabolism 
Description We have adapted our ultrafast time resolved fluorometer, which we originally developed for label-free readouts of tissue autofluorescence for clinical applications, and developed an instrument for measurements of plant autofluorescnce. We have shown that this can detect in vivo changes in plant metabolism foil lowing treatment with agrochemicals (specifically photosystem 2 inhibitors) and have deployed this to make measurements on living plants in the laboratory and in a green house 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2016 
Impact We have preliminary data indicating that we can distinguish the response of normal plants and plants with with chemical resistance. This could be important for in situ optimisation of the application of agrochemicals. 
 
Description Biophotonics Summer School Hven 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact I lectured on the Biophotonics Summer School organised in Hven, Sweden every two years. This is probably the highest level school int he field in terms of the lecturers and attracts students from all over the world. I presented the basic principles of fluorescence microscopy and our latest research in multidimensional fluorescence imaging including super-resolved microscopy, FLIM and optical tomography as well as clinical applications
Year(s) Of Engagement Activity 2015
URL http://www.biop.dk/biophotonics15/school/school.asp?page=main_school_lecturers
 
Description Conference presentation at Photon14 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Conference presentation at Photon14 - Fluorescence lifetime imaging endoscopes for biomedical applications - H. Sparks, I Munro, G Kennedy, E Hirata, S Warren, D Kelly, E Sahai, N Guerra, T Tatla, C Dunsby and P French
Year(s) Of Engagement Activity 2014
 
Description Invite presentation at the MDC Berlin 2015 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact High speed 2-D and 3-D fluorescence imaging of cardiomyocytes by light sheet microscopy and autofluorescence spectroscopy and imaging of biological tissue for clinical applications
C. Dunsby
Invited colloquium presentation at the MDC Berlin, February 2015
Year(s) Of Engagement Activity 2015
 
Description Invited conference presentation - Brighton 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Multi-dimensional fluorescence spectroscopy and imaging of tissue autofluorescence
C. Dunsby
Invited talk at the International Conference on Medical Physics, Brighton, 1st Sept 2013
Year(s) Of Engagement Activity 2013
 
Description Invited conferenence presentation - Imperial and KCL joing BHF Centres of Excellence Imaging Symposium London 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other audiences
Results and Impact Autofluorescence
C. Dunsby
Invited oral presentation at the Imperial College London and King's College London Joint BHF Centres of Research Excellence Imaging Symposium, London, June 2015
Year(s) Of Engagement Activity 2015
 
Description MDFI user-orientated workshop 2017 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact This one day user-orientated workshop entitled "Multidimensional fluorescence imaging technology: super-resolution, HCA and preclinical imaging" showcased the biophotonics instrumentation and applications of the technologies developed in the Photonics Group at Imperial College London. It attracted 80 participants including 11 from industry and 40 for other research organisation. The talks and posters were well received and this event led to further collaborations, including with industry.
Year(s) Of Engagement Activity 2017
 
Description MDFIM user-orientated workshop 2013 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Type Of Presentation Workshop Facilitator
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Multidimensional fluorescence imaging and metrology user-orientated workshop held at Physics Department, Imperial College London on 27/09/13.
~50 external visitors attended talks, posters and networking sessions. The aims were to identify new users of our research, to reinforce relationships with existing users, to identify new research partners, sponsors and opportunities. The event was also a showcase for our research progress and students and staff and a source of feedback to inform our ongoing technology development.

Three companies expressed interest in commercialising aspects of our work
Several external research partners learned about our capabilities and suggest new research collaborations
We received excellent feedback about quality of work presented and of presentations
Year(s) Of Engagement Activity 2013
URL http://www3.imperial.ac.uk/photonics/events/photworkshop
 
Description Oral presentation at BiOS 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Benjamin T. Dyer, J. Lagarto, M. B. Sikkel, C. B. Talbot, N. S. Peters, A. R. Lyon, P. M. French, C. Dunsby, "Application of autofluorescence lifetime metrology as a label-free technique to assess heart disease", BiOS 2014 SPIE Photonics West, San Francisco, California, United States, 2014. Oral presentation by Benjamin T. Dyer.
Year(s) Of Engagement Activity 2014
 
Description Oral presentation at BiOS 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact J. Lagarto, C. B. Talbot, B. Dyer, D. J. Kelly, M. Nickdel, M. B. Sikkel, J. Dudhia, Y. Itoh, N. S. Peters, A. R. Lyon, C. Dunsby, P. M. W. French, "A compact and portable hyperspectral autofluorescence lifetime point probe system applied to the study of cardiac disease and arthritis", BiOS 2014 SPIE Photonics West, San Francisco, California, United States, 2014. Oral presentation by J. Lagarto.
Year(s) Of Engagement Activity 2014
 
Description Oral presentation at BiOS 2014 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Development and characterisation of fluorescence lifetime imaging endoscopes for biomedical applications
H. Sparks, I. Munro, G. Kennedy, E. Hirata, E. Nigar, S. Warren, E. Sahai, T. Tatla, C. Dunsby and P. M. W. French
Oral presentation at BiOS, Photonics West, San Francisco, 2014
Year(s) Of Engagement Activity 2014
 
Description Oral presentation at Laser Europe 2014 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other audiences
Results and Impact J. Lagarto, C. B. Talbot, B. Dyer, D. J. Kelly, M. Nickdel, M. B. Sikkel, J. Dudhia, Y. Itoh, N. S. Peters, A. R. Lyon, C. Dunsby, P. M. W. French, "A compact and portable hyperspectral autofluorescence lifetime point probe system applied to the study of cardiac disease and arthritis", Laser Europe 2014, Amsterdam, The Netherlands, 2014. Oral presentation by J. Lagarto.
Year(s) Of Engagement Activity 2014
 
Description Photon 14 conference presentation 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Postgraduate students
Results and Impact Conference presentation at Photon 14 by Joao Lagarto - Compact and portable hyperspectral autofluorescence lifetime point probe system applied to the study of disease - J. Lagarto, C. B. Talbot, B. Dyer, D. J. Kelly, M. Nickdel, M. B. Sikkel, J. Dudhia, Y. Itoh, N. S. Peters, A. R. Lyon, C. Dunsby, P. M. W. French
Year(s) Of Engagement Activity 2014
 
Description Photonics Group evening workshop on clinical applications of fluorescence spectroscopy and imaging 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Professional Practitioners
Results and Impact Meeting of current and potential clinical collaborators to discuss the clinical application of the Photonics Group's fluorescence spectroscopy and imaging technologies. Attended by 10 clinicians, one member of industry and the project team (10 members).

The meeting provided an opportunity to disseminate the group's work to a focused clinical audience and stimulated useful debate about current work and future directions. This meeting helped stimulate a number of future potential research directions.
Year(s) Of Engagement Activity 2015