Endoscopic FLIM for label-free tissue contrast

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

Abstract

This project aims to provide new label-free imaging tools for minimally invasive diagnosis of diseases including cancer. When irradiating tissue with light at an appropriate wavelength, many molecules absorb this excitation energy and emit new radiation called fluorescence . As these fluorescent molecules occur naturally in biological tissue, their emission is called autofluorescence. By analysing such autofluorescence signals, it is possible to detect the presence of particular kinds of molecule, e.g. providing information on the tissue structure, and also to learn about their local environment, e.g. whether they are bound to other molecules. Autofluorescence may therefore provide a means to detect the early onset of diseases that cause changes in the concentration, distribution and interaction of biological molecules. Because autofluorescence measurements do not require the addition of any chemicals, this approach is label-free and can be non-invasive, making it attractive for diagnostic applications. Biological tissue, however, often contains several kinds of fluorescent molecule in unknown quantities and strongly scatters optical radiation, making quantitative fluorescence measurements difficult. It is therefore desirable to analyse tissue autofluorescence in a way that avoids intensity artefacts and to acquire images so that variations in the autofluorescence signal can be correlated with the observed structures in the tissue. This is analogous to conventional histopathology, where diagnoses are made following biopsy using images of sections of biological tissue that have been stained with dyes to indicate the distributions of different types of molecule. In this proposed work, we will develop a novel endoscope to provide microscope-like images of biological tissue with the autofluorescence signal providing molecular contrast. To quantify the autofluorescence signal, we will exploit the fact that different molecular species radiate fluorescence at different rates and so it is possible to distinguish them by observing the fluorescence decay times (lifetimes) for each pixel in the field of view. By combining fluorescence lifetime imaging (FLIM) with a special endoscope that provides microscope images with depth resolution, we will be able to perform optical biopsy in situ, analysing optically sectioned images with fluorescence lifetime providing the molecular contrast.We have investigated FLIM of biological tissue since 1998, demonstrating some of the first label-free lifetime contrast of ex vivo disease in tissue (e.g. cancer, osteoarthritis and atherosclerosis) and have developed a range of sophisticated laboratory-based FLIM instrumentation including proof-of-principle FLIM endoscopy. It is now vital to progress to in vivo imaging using a clinically viable endoscope-based approach. The world-leading endoscopic confocal microscope developed by Mauna Kea Technologies (MKT) provides optically sectioned imaging with subcellular resolution and has been approved for clinical use in Europe and the USA. Currently it is limited to intensity imaging at a single excitation wavelength (488 nm). We aim to develop a FLIM version of this endoscope, initially using 488 nm excitation and then expanding to shorter wavelengths in order to excite more biological molecules. This instrument will be highly suitable for optical biopsy but the inherently limited field of view of confocal endoscopy will limit its application for screening of disease. We will therefore also develop a clinically viable wide-field FLIM endoscope to provide a larger viewing area, albeit without optical sectioning. This will permit a comparison of the performance of these two approaches to endoscopy. We will also correlate endoscopic FLIM with histopathology and with existing advanced FLIM instrumentation at Imperial, which will help elucidate the molecular origins of the autofluorescence contrast we observe between normal and diseased tissue.

Publications

10 25 50
 
Description We applied the MKT Cellvisio™ confocal endomicroscope to image tissue autofluorescence excited at 488 nm, mainly imaging elastin in lung tissue, and developed it for real-time (~Hz) FLIM using TCSPC. Unfortunately, our spectral characterisation of its performance revealed that tissue autofluorescence was swamped by background fluorescence in the fibre bundle at the desired excitation wavelengths. Working with MKT we evaluated a new lower background fibre bundle and determined that 420 nm excitation maximised autofluorescence signal/fibre bundle background but could not achieve sufficient performance for clinical applications. Accordingly we changed our objectives (agreed during EPSRC review visit 03/11/09) and redeveloped this instrument for tissue imaging with exogenous probes. With St Mary's Hospital, we are investigating its potential to image PpIX in the bladder for cancer detection and explored new excitation wavelengths optimising PpIX signal/fibre bundle background. We also collaborated with CRUK to demonstrate imaging of CFP/YFP/GFP labelled tissue ex vivo and are now working towards FLIM FRET endomicroscopy inside live mice.

We undertook extensive spectrally and lifetime resolved ex vivo studies of a range of tissue types (colon cancer, Barrett's oesophagus, adenomas and inflammatory bowel disease). In a study of 65 specimens from 30 patients we observed statistically significant lifetime contrast between normal and dysplastic or inflammatory tissues. We also employed our SAFE probe to study ex vivo GI tissue and showed statistically significant contrast between normal colon tissue relative to polyps and polyps relative to irritable bowel disease (inflammation), which is clinically useful. Initial studies also indicate contrast between normal colon and inflammatory tissue.

We redesigned our clinical FLIM programme, also taking into account the late recruitment of our clinical research fellow (after a previous applicant withdrew at a late stage). To meet our main priority of comparing ex vivo and in vivo autofluorescence lifetime signatures of disease, we switched from the MKT system to the SAFE probe and undertook two clinical trials. The first study was of skin cancer in 25 patients and we demonstrated significant lifetime contrast for blue excitation, in agreement with ex vivo studies. The second study used the SAFE probe to measure GI tissue in 17 patients in vivo, with initial data indicating blue excitation provides contrast broadly consistent with ex vivo studies. In addition, we applied a wide-field FLIM instrument applied to freshly excised ENT tissue, which will be translated to an in vivo clinical trial using our newly designed wide-field flexible FLIM endoscope once we have the regulatory approval.

We developed a new concept for ultracompact endoscopy (patent application filed) that utilises multiphoton excitation to minimise background (fibre bundle) fluorescence and, uniquely, eliminates unwanted nonlinear optical effects by distributing the excitation power across 100's of cores in a fibre bundle and uses adaptive optics to control the phase of the distal beams such that they can be scanned and focussed with no optical or mechanical components. This permits 3-D imaging via ultrathin (~100's microns) endoscopes - potentially providing unprecedented access to internal tissues. We are now working towards a clinical demonstrator.
Exploitation Route As discussed above, the instrumentation we have developed could be used in further clinical trials where it is close to providing useful diagnostic information for clinical screening, e.g. in the GI tract as part of routine endoscopic investigations, and for defining tumour margins during surgery, e.g. in the throat (around vocal chords) and in the brain. It could also be used to assist Moh's procedure. A further application is in monitoring organ viability for transplant patients and we are in discussion with the Chelsea and Westminster hospital to explore this potential for kidney transplants.

The instrumentation developed in this project could also be used for animal studies, including veterinary applications, and potentially for biotechnology including for real-time monitors of tissue engineering bioreactors.

Our goal is to develop clinical instrumentation to provide label-free diagnosis and monitoring of disease. A key achievement for this project has been to undertake clinical trials and to confirm that the autofluorescence lifetime signatures that we see ex vivo are present in vivo. We have realised this goal to some extent but further trials are needed to establish the clinical efficacy of autofluorescence lifetime readouts and this is a key strand of our exploitation strategy. To this end we are currently conducting an ex vivo study at Northwick Park hospital exploring ENT applications using wide-field FLIM instrumentation developed during this project and are setting up an in vivo clinical trial in this area, to be conducted by an EPSRC DTG-funded PhD student who is completing the fabrication of a new wide-field flexible FLIM endoscope intended for this trial. We will work with our partners at Charing Cross, Hammersmith, Northwick Park and St Mary's hospitals to identify and address further opportunities for clinical trials and to refine our FLIM endoscopes for maximum clinical utility. Our clinical research fellow (whose clinical PhD work within this project won two international prizes) has applied for an EPSRC Doctoral Prize award to continue this vital clinical imaging with the existing instrumentation developed under this project. Besides PhD students we will be seeking resources from NIHR and the MRC to continue this work. The single point autofluorescence endoscopic probe is also being applied to explore its potential for studying heart disease and osteoarthritis as part of an ongoing EPSRC Healthcare partnership award. We would also like to support ongoing endoscope development through an EPSRC Platform Grant, for which we are finalising a proposal.

For optically sectioning high resolution endoscopy, we are working with the CRUK London Research Institute to address in vivo studies of live disease models for research and drug discovery. For this we are preparing to conduct the first in vivo experiments for applying the MKT confocal FLIM endomicroscope in mice labelled with genetically expressed fluorescent proteins, having already demonstrated the proof of concept in ex vivo mouse tissue. This would permit FLIM readouts of signalling processes in internal organs with sub-cellular resolution for the first time - thereby highlighting new market opportunities for MKT, for which we will work with Imperial Innovations to exploit this commercial development.



We are also working to exploit high resolution optical sectioning endomicroscopy for clinical applications. We have two strategies to address the limiting issue of background fluorescence in fibre-bundle endomicroscopes. For the MKT system, we aim to work with exogenous fluorophores excited at longer wavelengths and have explicitly investigated the imaging of PpIX fluorescence induced by administration of ALA-5. We will work to investigate this with fresh tissue resections of bladder tissue with St Mary's hospital, continuing our ongoing partnership. For imaging tissue autofluorescence, we are working to exploit our novel (patent application filed) endoscope concept utilising adaptive optics to realise ultracompact multiphoton endoscopes with no requirement for distal optics, mechanics or moving parts. We have applied for EPSRC Healthcare Engineering funding to translate the proof of concept demonstrated in this project to a clinical prototype. We will also look for other sources of technology transfer/follow-on funding, including from the Wellcome Trust and the Royal Society.

While our current FLIM endoscope technology is clinically deployable, their relatively high cost (£50k-200k for single-point/imaging systems) can ultimately limit their clinical deployment. We therefore initiated a programme within this project to develop more compact and lower cost single point fibre-optic probe-based instruments to measure the lifetime and spectral properties of (tissue) fluorescence using modulated diode lasers and ultrafast detection implemented in programmable FPGA electronic circuitry. This has the potential to significantly reduce the cost and the size of our instrumentation. Once we have a working prototype, we would evaluate it within the scope of our EPSRC Healthcare Partnership grant and seek an industrial partner to manufacture a number of such devices for multiple parallel clinical trials across a range of diseases and clinical sites. We already have an interested SME contributing the design of the electronics and we will seek to fund this development through technology transfer and/or TSB and NIHR funding
Sectors Healthcare

URL http://www.imperial.ac.uk/photonics/research/biophotonics/instruments--software/
 
Description This project demonstrated the feasibility of endoscopic FLIM and led to further projects including a PhD project to develop new FLIM endoscopes and two EPSRC funded projects that produced new medical instrument prototypes and trained further PhD students. The confocal FLIM endomicroscope has been applied to an in vivo study of drug-target engagement at the Francis Crick Institute, where we have established a satellite laboratory, partly to transfer the expertise and instrumentation specifically developed during this project.
First Year Of Impact 2010
Sector Education
Impact Types Societal

 
Description EPSRC Healthcare Partnership EP/I02770X/1
Amount £1,174,248 (GBP)
Funding ID EP/I02770X/1 
Organisation Engineering and Physical Sciences Research Council (EPSRC) 
Sector Public
Country United Kingdom
Start 07/2011 
End 03/2014
 
Description Fluorescence lifetime imaging endoscopy for PPIX detection
Amount £93,749 (GBP)
Funding ID II-3A-0409-10044 
Organisation National Institute for Health Research 
Sector Public
Country United Kingdom
Start 06/2010 
End 05/2011
 
Description Visiting Professorship
Amount £18,695 (GBP)
Funding ID F07058BH 
Organisation The Leverhulme Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 08/2009 
End 08/2011
 
Title Sparks Et Al, Heterogeneity In Tumor Chromatin-Doxorubicin Binding Revealed By In Vivo Fluorescence Lifetime Imaging Confocal Endomicroscopy: In Vitro Data 
Description Data is divided into three folders: Sparks_et_al_FIG2_Histone_vs_free_GFP data for Sparks et al Figure 2 main text section: 'FRET between  chromatin-bound GFP and doxorubicin' Sparks_et_al_FIG3_in_vitro_dose_response data for Sparks et al Figure 3# main text section: 'FLIM endomicroscope can monitor doxorubicin cellular uptake' Sparks_et_al_SuppFIG2_endoscope_spectral_cross_talk data for Sparks et al Supplementary Figure 2 Supplementary information Cell lines IGROV-1 cell lines were cultured in CO 2 dependent media with 10% fetal bovine serum and 1% Pen Strep at 37 ?C. Before experiments, cells were grown to 80% confluence. For measuring doxorubicin uptake by fluorescence an IGROV-1 cell line stably expressing GFP fused to Histone-1 (H1) was made using the PiggyBac transposon system. As a control to show that effect of doxorubicin on GFP depends on whether it is fused to H1 or not, a stable whole cell expression of GFP by lentiviral transfection and selection by Geneticin was made. For bioluminescence imaging of xenograft tumors, all IGROV-1 cell lines were made to stably express firefly luciferase. To investigate the effect of doxorubicin on other histones, IGROV-1 cells were transiently transfected with a Histone-2B-GFP plasmid (gift from Kurt Anderson) using the Lipofectamine® 2000 reagent. IGROV-1 cells were obtained from Crick institute cell services and confirmed as IGROV-1 by Short Tandem Repeats (STR) profiling and no mycoplasma was detected. In vitro experiments IGROV-1 cells were grown to 80% confluence in 75 ml flasks before being re-plated in 12 or 24 well plates or 35 ml glass bottomed dishes and allowed to attach to the surface for 24 hours before experiments. To study how the fluorescence of GFP labelled H1 labelled IGROV-1 cells changes with doxorubicin treatment, fluorescence intensity and lifetime distributions were measured from cells after 3 hours of incubation with doxorubicin of varying concentrations (0, 0.18, 0.9, 1.8, 9, 18 µM) by serial dilutions of a stock solution with PBS. After 3 of hours, cells were washed in PBS then fixed for 20 minutes in 4% PFA. Cells were then imaged in PBS.  Doxorubicin hydrochloride (Sigma-Aldrich, D1515-10 mg) was dissolved in PBS to a concentration of 9 mM and stored at -20?C. 
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
 
Title Sparks Et Al, Heterogeneity In Tumor Chromatin-Doxorubicin Binding Revealed By In Vivo Fluorescence Lifetime Imaging Confocal Endomicroscopy: In Vivo Data 
Description Data is divided into three folders: Sparks_et_al_FIG_6_IP_intranodule_heterogeneity data for Sparks et al Figure 6 main text section: 'FRET between  chromatin-bound GFP and doxorubicin' Sparks_et_al_FIG4_5_6_IP_IV_chemo_comparison data for Sparks et al Figures 4,5 & 6 main text section: 'FLIM endomicroscope can monitor doxorubicin cellular uptake' Sparks_et_al_FIG6_IP__internodule_heterogeneity data for Sparks et al Figure 6 main text section: 'Intra-tumor heterogeneity' In vivo experiments Murine xenografts were prepared by intraperitoneal (IP) injection of IGROV-1 cancer cells. IGROV-1 cells were grown to 80% confluence before being trypsinized and re?suspended in PBS at a concentration of  cells per ml.  cells were injected into ICRF nude mice. After 14 days post-injection, the presence of intraperitoneal tumors was confirmed by bioluminescence imaging. Briefly, an IVIS bioluminescence imaging system was used to image isoflurane anesthetized mice. 100 µl of D-luciferin (luciferase substrate) at 30mg ml -1 was injected IP 10 minutes before recording of bioluminescence images. The presence of peritoneal tumors was confirmed if bioluminescence signals from the peritoneum were above background noise 10-30 minutes after D?luciferin injections. Following confirmation of tumors, in vivo fluorescence imaging experiments were carried out after 21 days. To study differences in drug uptake between intravenous or intraperitoneal delivery, prior to imaging mice were subject to IP or IV doxorubicin-based chemotherapy for 1.5, 3 or 24 hours. Imaging involved terminal procedures, mice were anesthetized then peritoneal tumors were exposed by minor surgery and inspected with the CEM. All animal model procedures were approved by The Francis Crick Institute Biological Ethics Committee and UK Home Office authority provided by Project License 70/8380.     
Type Of Material Database/Collection of data 
Year Produced 2018 
Provided To Others? Yes  
 
Description Cancer Research UK, London Research Institute 
Organisation Cancer Research UK
Department Cancer Research UK London Research Institute (LRI)
Country United Kingdom 
Sector Academic/University 
PI Contribution Research Institute. We have worked with Erik Sahai?s group at CRUK-LRI to demonstrate the capabilities of our new confocal FLIM endomicroscope, demonstrating its ability to obtain fluorescence lifetime images of ex vivo mouse tissue expressing CFP, YFP, GFP and also treated with drugs. We are working towards the first demonstration of in vivo FLIM endoscopy with this endomicroscope aiming to readout FRET of signalling molecules.
Start Year 2011
 
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
 
Description Mauna Kea Technologies, SA 
Organisation Mauna Kea Technologies
Country France 
Sector Private 
PI Contribution Collaboration to develop FLIM endomicroscopes. We adapted aCellvisio endomicrocope to FLIM using TCSPC and evalauted it for iamging tiissue and fluorescently labelled biological samples.
Collaborator Contribution Mauna Kea Technologies provided us with an endoscope system at significant discount and the expertise and confidential information to enable us to develop a FLIM endomicroscope using their Cellvisio platform
Impact We developed a FLIM confocal end microscope system that we have applied to FRET of cells expressing biosensors and recently translated to in vivo preclinical imaging
Start Year 2008
 
Description Northwick Park Hospital NHS Trust 
Organisation Northwick Park Hospital
Country United Kingdom 
Sector Hospitals 
PI Contribution Taranjit Tatla, ENT surgeon. Taranjit Tatla is working with us to explore clinical opportunities for FLIM endoscopy. We are currently collaborating on FLIM of ex vivo tissue using our instrumentation adjacent to the operating theatres at Northwick Park hospital to determine the optimum parameters for our wide-field FLIM endoscope and are preparing to apply to approval to conduct an in vivo clinical trial imaging ENT cancer.
Collaborator Contribution Taranjit Tatla worked with us to evaluate the efficacy of our wide-field FLIM instrument on ex vivo tissue at Northwick Park hospital and helped us interpret the data from a clinical perspective.
Impact Taranjit Tatla included this work in his PhD thesis and presented results at a clinical conference. This was a multidisciplinary collaboration between Physics and Medicine.
Start Year 2011
 
Title IMPROVED ENDOSCOPE 
Description Novel approach to realising laser scanning endoscopy that can be used to realise thinner endoscopes. Also useful for multiphoton endoscopy as it reduces peak power of excitation pulses in optical fibres. 
IP Reference WO2010004297 
Protection Patent application published
Year Protection Granted 2008
Licensed No
Impact Publications and a new EPSRC research grant
 
Title Adaptive optics multiphoton endomicroscope 
Description We have developed a new concept for ultracompact 3-D endomicroscopy (patent application filed) that utilises multiphoton excitation to minimise background (fibre bundle) fluorescence and, uniquely, eliminates unwanted nonlinear optical effects by distributing the excitation power across 100?s of cores in a fibre-optic bundle and uses adaptive optics to control the phase of the distal beams emerging from the optical fibre cores such that they can be scanned and focussed with no optical or mechanical components. This new approach permits 3-D imaging via ultrathin (~100?s microns) fibre-optic bundles with no need for distal optics or moving parts since both scanning and focussing can be implemented by manipulating the phase profile of the excitation light emerging from the optical fibre cores at the distal end of the fibre optic bundle. This could potentially provide unprecedented access to internal tissues. We have experimentally demonstrated the proof of concept and are working to demonstrate the first prototype instrument in order to proceed to a clinical demonstration. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2011 
Impact New EPSRC grant to further develop device 
 
Title Confocal FLIM endomicroscope 
Description We developed the first confocal FLIM endomicroscope, providing optically sectioned fluorescence lifetime images with subcellular resolution for the first time via an endoscope. This was based on the Mauna Kea Technologies Cellvisio? system, which we developed into a self-contained trolley based instrument incorporating a tunable ultrafast excitation laser (based on a frequency-doubled Ti:Sapphire laser) and implemented time-correlated single photon counting (TCSPC) detection. We wrote the acquisition and analysis software and demonstrated the capability to image (mouse) tissue expressing CFY, YFP and GFFP in real-time (~Hz). This confocal FLIM endomicroscope can be configured with outside diameters of less than 2 mm, providing access to many internal organs in humans and animals. For animal imaging the is tremendous potential impact for drug discovery, preclinical imaging and basic biomedical research if cell signalling processes could be read out in real time in live disease models using Forster resonant energy transfer (FRET) to study protein-protein interactions. To this end we showed that our instrument can realise FLIM FRET of live cells in expressing GFP and mCherry constructs and we are working with the CRUL London Research Institute to translate this to in vivo experiments. We are also working to apply this to clinical imaging of cancer with exogenous probes such as PPIX induced by ALA-5, for which we have an ongoing collaboration with St Mary?s Hospital. We extensively evaluated this instrument for label-free imaging of tissue autofluorescence but unfortunately fluorescence is excited in the glass fibre bundles at the same wavelengths as biological tissue is effectively excited for clinical readouts (i.e. in the u.v./blue) and the unwanted background fluorescence from the fibre-optic bundle swamps the tissue autofluorescence. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2010 
Impact This endoscope has the potential to enable FLIM to be applied in vivo, including in animal models. This would permit minimally invasive measurements of, e.g. protein-protein interaction,s that could provide information about disease processes or the effects of therapeutic intervention and would require fewer animals for such studies compared to invasive surgery. 
 
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 Wide-field clinical FLIM platform 
Description We have also developed our wide-field FLIM instrumentation for in situ clinical investigations in hospitals by developing a self-contained trolley-based platform for both macroscopic imaging of larger (cm scale) tissue resections that we can obtain straight from the operating theatre before they are sent for histopathological analysis. For in vivo application we have developed the world?s first handheld wide-field FLIM system, which integrates a low cost CCD with a gated image intensifier in a compact pistol-grip until that can be held by a clinician and optically coupled to rigid or flexible endoscopes where necessary. We are also finalising a prototype wide-field flexible FLIM endoscope for clinical imaging of tissue autofluorescence that overcomes the problem of unwanted background fluorescence from the optical fibre bundle and that can be sterilised such that it can be used in vivo. This instrument will have a relatively large (~cm) field of view potentially making it suitable for diagnostic screening (using FLIM to provide a red flag technique, e.g. to guide biopsies) and for assessing tumour margins. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2010 
Impact This work gave us and the international community insights into how fluorescence lifetime can report on cancerous tissue but also on how the signature can vary according to the specific pathology 
 
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 EPSRC MRC Medical Imaging Technology Working Group 
Form Of Engagement Activity A formal working group, expert panel or dialogue
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Policymakers/politicians
Results and Impact Paul French served on this EPSRC/MRC panel to review funding in medical imaging Paul French served on this EPSRC/MRC panel to review funding in medical imaging research and to advise on future strategy and priorities. Awarding Body - EPSRC, MRC, Name of Scheme - Medical Imaging Technology Working Group
Year(s) Of Engagement Activity 2011
 
Description Fluorescence lifetime imaging - for cell biology, drug discovery and clinical diagnosis 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact PicoQuant GmbH 4th European Short Course on "Time-resolved Microscopy and Correlation Spectroscopy", Berlin, Germany.
Year(s) Of Engagement Activity 2012
 
Description GALDERMA 2009 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Industrial visit and seminar.

Paul French was invited to present our work on tissue FLIM to this dermatology company in their headquarters near Nice with a view to exploring possible collaborations.
Year(s) Of Engagement Activity 2009
 
Description GSK Stevenage 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Industry/Business
Results and Impact Industry visit and seminar.

Paul French gave an invited seminar on FLIM and its biomedical applications including results from this project.
Year(s) Of Engagement Activity 2012
 
Description International School of Physics "Enrico Fermi": "Microscopy Applied To Biophotonics" 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited lectures at the International School of Physics "Enrico Fermi": "Microscopy Applied To Biophotonics", Varenna, Lake Como, Italy, 2011



Introduction to the sources of cellular and tissue autofluorescence; Microspectrophotometry and imaging for studying celluar autofluorescence in vitro; & Autofluorescence instrumentation.
Year(s) Of Engagement Activity 2011
 
Description Karl Storz GmbH, Tuttlingen 2008 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Industry/Business
Results and Impact Industry visit and seminar.

Paul French was invited to present our work on tissue FLIM to this endoscopy company with a view to exploring possible collaborations. This led to an NIHR grant to investigate the potential for FLIM endoscopy of PPIX fluorescence as an indicator of bladder cancer
Year(s) Of Engagement Activity 2008
 
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 Multidimensional fluorescence imaging 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience
Results and Impact Invited talk, Practical course: Monitoring biochemical interactions in living cells, DKFZ, Heidelberg, Germany.
Year(s) Of Engagement Activity 2008
 
Description Multidimensional fluorescence imaging 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience
Results and Impact Invited talk, Summer School, KSOP, Karlsruhe, Germany.
Year(s) Of Engagement Activity 2008
 
Description Multidimensional fluorescence imaging 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience
Results and Impact Leica Scientific Forum Lecture tour: Fresnel Institute/Marseille; EMBL/Heidelberg;

Curie Institute/Paris.
Year(s) Of Engagement Activity 2008
 
Description Multidimensional fluorescence imaging 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience
Results and Impact Invited talk, FEBS Summer School, Wageningen, Netherlands.
Year(s) Of Engagement Activity 2008
 
Description Multidimensional fluorescence imaging 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience
Results and Impact Invited talk, 1st Short Course on Time-Resolved Microscopy & Correlation Spectroscopy, PicoQuant GmbH, Berlin, Germany.
Year(s) Of Engagement Activity 2009
 
Description Multidimensional fluorescence imaging - for clinical diagnosis, cell biology and drug discovery 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact 1st Europhotonics Spring School, Barcelona, Spain.
Year(s) Of Engagement Activity 2012
 
Description Multidimensional fluorescence imaging and metrology - for cell biology, high content analysis and clinical diagnosis 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited lectures at Biophotonics 2011 summer school, Ven, Sweden.
Year(s) Of Engagement Activity 2011
 
Description Multidimensional fluorescence imaging for cell biology, high content analysis and label-free diagnosis 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience
Results and Impact Invited lecture at FEBS Summer School, Wageningen, The Netherlands.
Year(s) Of Engagement Activity 2010
 
Description Multidimesional fluorescence imaging 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience
Results and Impact Invited oral presentation at the 2nd European Short Course on Time-Resolved Microscopy and Correlation Spectroscopy, Berlin, Germany.
Year(s) Of Engagement Activity 2010
 
Description Mutidimensional fluorescence Imaging 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited lectures, Fourth International summer school, Nanosciences Ile de France, France.
Year(s) Of Engagement Activity 2010
 
Description PQ Short course MDFI 2008 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited talk, 6th European Short Course on Principles and Applications of Time-resolved fluorescence, PicoQuant GmbH, Berlin, Germany.
Year(s) Of Engagement Activity 2008
 
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
 
Description PicoQuant workshop 2013 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Postgraduate students
Results and Impact Invited oral presentation at the 3rd European Short Course on Time-Resolved Microscopy and Correlation Spectroscopy, Berlin, Germany. mainly aimed at post-graduate students

Pau French was asked to come back again to lecture on this training course
Year(s) Of Engagement Activity 2012
 
Description SPIE Newsroom news article, July 2008 FLIM for drug discovery and disease research 
Form Of Engagement Activity A magazine, newsletter or online publication
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Industry/Business
Results and Impact SPIE Newsroom news article, July 2008.
Year(s) Of Engagement Activity 2008