Development of multiphoton microscopes for real-world clinical applications

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


At present, the diagnosis of many types of disease must be confirmed by histopathology before treatment can begin. This entails the physical removal of a small tissue specimen from the patient that is then processed, sliced, stained and analysed by an expert using an optical microscope. The collection of tissue is subject to sampling error, i.e. the diseased tissue may be missed, and the time required for processing and analysis can delay treatment. It would desirable for physicians to be able to make an immediate diagnosis during examination of the patient.

When biological tissue is illuminated with light at appropriate wavelengths, certain naturally occurring biomolecules can absorb this excitation energy and emit new light called autofluorescence. Normally each absorbed photon results in a photon being emitted at longer wavelength (lower photon energy). This can be used to provide high resolution images of biological tissue and being "label-free", it avoids potential hazards associated with externally applied contrast agents. The properties of this light can provide information about the molecules that emitted it and this enables imaging of the biochemical or structural changes in tissue associated with disease, which can aid diagnosis. As well as the wavelength (colour) of the autofluorescence, the fluorescence lifetime (i.e. the rate at which the signal decays following excitation) can also provide useful "spectroscopic" information.

A key challenge for optical imaging in tissue, however, is optical scattering, which limits the depth at which optical images can be obtained. This can be addressed using the technique of multiphoton microscopy, which entails illuminating tissue with light at twice the wavelength (half the photon energy) that is usually absorbed. If the incident intensity is high enough, then the tissue can absorb two photons simultaneously to produce each autofluorescence photon. This permits excitation of tissue at longer wavelengths (which undergo less scattering and permit deeper imaging) and also provides 3D imaging. This is because the autofluorescence photons are only efficiently produced in the focal plane where the intensity is highest. Thus a multiphoton microscope provides "optical sectioned" images of slices of tissue that are in the focal plane. Currently, however, there is only one commercially available clinically approved multiphoton microscope, which is licensed for imaging skin, and no clinical multiphoton endoscope. We propose here to develop two new complementary approaches to in vivo clinical multiphoton imaging that should enable high resolution (subcellular) imaging of less accessible (including internal) tissues to provide real-time replacements for conventional histopathology - allowing in situ diagnosis and guiding of therapeutic interventions where it is critical to define lesion margins and minimise loss of function, e.g. in the brain, prostate, spine, skin (Moh's surgery) etc.

The first instrument is a novel lightweight hand-held multiphoton scanner, which would be able to compensate for patient motion (permitting longer acquisition times), richer spectroscopic readouts (including of fluorescence lifetime) and larger fields of view (to permit visualisation of whole lesions). This instrument would be applicable to image any external tissue or to tissues exposed during surgery.

The second instrument is a disruptive technology concept invented and pioneered at Imperial for ultracompact multiphoton endoscopes of unprecedented size (<400 microns diameter) and flexibility, for use via fine needles directly in the organ of interest or via thin anatomical channels (down to breast ducts). It could thus provide sub-cellular imaging almost anywhere inside the body, including under the guidance of other imaging modalities (e.g. ultrasound, MRI).

These instruments would be engineered for clinical trials following ex vivo human tissue studies during this project.

Planned Impact

This project aims to provide new non-invasive tools for improved patient diagnosis and treatment and for improved capabilities for in vivo research. Thus this research should ultimately benefit patients and reduce costs to the NHS and other healthcare providers by providing faster optical readouts of tissue state ("optical biopsy"), by allowing a greater number of tissue sites to be probed in a clinical examination, by reducing the need for tissue biopsies and by enabling imaging of sensitive areas of tissue that cannot be biopsied. Examples include reducing or replacing the need for rapid cryo-histology during tissue-sparing surgery such as neurosurgery or Moh's surgery, thereby enabling these procedures to become faster and more accurate - hence improving patient outcomes. The proposed new instrumentation would also 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 metabolism or tissue matrix properties are useful.

In vivo imaging technologies are also important for drug discovery and therefore the pharmaceutical industry, e.g. during preclinical drug testing in animals, and would permit longitudinal studies, therefore reducing the required numbers of animals and increasing the value of the data. Their application with fluorescence labels, including fluorescent proteins in genetically manipulated live disease models, could provide in vivo readouts of signal pathways for drug discovery and the label-free readouts based on autofluorescence could provide information on changes in metabolism and tissue matrix properties. Importantly, label-free readouts could be translated directly from animals to humans and used to evaluate new therapies, including drugs, and to monitor therapeutic procedures and outcomes in patients.

The most immediate impact of this project would be the subsequent medical research utilising the label-free detection, diagnosis and monitoring of disease afforded by the new instruments in the areas of our clinical advisory panel members, which include dermatology, gastrointestinal endoscopy, head and neck surgery and neurosurgery. We thus envisage patient-orientated follow-on studies and clinical trials to establish the patient benefit and/or cost-savings of these new clinical tools. For neurosurgery there is a specific unmet need for a minimally invasive functional and spatially-resolved readout of brain tissues that allows surgeons to decide whether it is necessary to perform further excisions and this would be one of our first targets.

Commercialisation would be an important route to realising the anticipated biomedical impact and this project would also stimulate the development of related instruments optimised for a range of applications, including with lower cost laser sources, thereby benefitting the instrumentation industry and the economy. We are seeking commercial exploitation partners and want to use the data from this project to build the prototypes needed to carry out clinical trials of the technology that are needed to validate the clinical applicability and strengthen our case for commercial investment. We already have strong links with biomedical instrument manufacturers including Mauna Kea Technologies (MKT, France) and JenLab (Germany) who are current market leaders in the field of in vivo clinical (endo)microscopy and note that the chief scientific officer of MKT will sit on our clinical advisory panel. MKT have already expressed interest the adaptive MP endoscopy technology, which could complement their existing clinically licensed endomicroscopes. Any exploitation of existing IP would be managed by Imperial's tech transfer company Imperial Innovations, in collaboration with Bath Ventures.

We also note the valuable training of project research staff who will gain interdisciplinary training at the interface between physics and medicine.
Description During this research project we developed two novel multiphoton-excited fluorescence imaging instruments.

The first instrument is a handheld multiphoton microscope with built-in 3-D motion compensation developed for skin imaging with sub-cellular spatial resolution. A key novel aspect of this instrument is the use of optical coherence tomography to track the surface of the skin to enable active axial motion compensation, such that the desired image plane in the tissue remains in focus even if the distance between the sample and handheld scanner changes. This enables our device to image in vivo with subcellular resolution even in parts of the body affected by motion, e.g. due to normal breathing. The performance of this handheld multiphoton microscope was successfully tested by imaging human chest skin in vivo with the volunteer sitting and the instrument hand-held. We have also extended this approach to provide a novel method for lateral motion compensation. This was also demonstrated in vivo has been published.

Working with the University of Bath, we tested the use of a new class of optical fibres - called negative curvature fibres - and demonstrated for the first time that they can be used to couple ultrafast laser radiation to the handheld scanner with unprecedented preservation of the peak pulse power to enable multiphoton microscopy. These fibres allow excitation radiation to be delivered over a wide range of wavelengths with low attenuation and without causing nonlinear spectral or temporal broadening of the ultrashort pulses - prior to this, it has not been possible to achieve all of these desirable features at the same time.

The second instrument is an adaptive multiphoton endoscope employing a custom "bundle" of 96 single-mode polarisation optical fibres developed by the University of Bath. This multiphoton-excited fluorescence endoscope requires no optical or mechanical components at the distal end to achieve scanning and focussing of the excitation beam, enabling ultranarrow endoscopes to be developed that could access hitherto unreachable regions of a patient. We published the first report of an adaptive multiphoton endoscopy using a polarisation-maintaining multicore fibre and we analysed the theoretical gain in performance this fibre offers over non-polarisation maintaining fibre bundles. Excitingly, we have also demonstrated the first imaging through such a microendoscope while the optical fibre is being flexed and the results have been published.

The University of Bath also fabricated a number of novel multicore optical fibre designs aiming to increase the number of image resolution elements of the endoscope, the field of view and the contrast to background of the imaging process. These optical fibres were successfully tested and comprehensively evaluated at Imperial and these results published in a peer-reviewed journal.
Exploitation Route For the handheld multiphoton scanner, we have demonstrated novel experimental designs for both axial and lateral image stabilisation to account for sample motion during image acquisition and we believe that these developments have advanced the state of the art in this field. In the future we anticipate that these developments will be used by others to improve the imaging performance in a range of preclinical and clinical multiphoton imaging systems. We also demonstrated the use of negative curvature fibres for delivering ultrafast pulsed radiation for multiphoton microscopy for the first time. These fibres allow tunable ultrashort pulse radiation to be delivered over a broad range of wavelengths with low attenuation and without nonlinear spectral or temporal broadening, which we believe will be of interest for a range of applications, particularly those utilising nonlinear optics or ultrafast light-matter interactions.

For the adaptive endoscope, this project has developed a number of novel multi-core single mode optical fibre designs that have enhanced the state of the art of the field of adaptive multiphoton endoscopy. We have also published a theoretical analysis of the benefits of using polarisation maintaining fibre bundles for this application. We believe that these results will provide valuable information to inform this rapidly emerging field and speed the path towards devices that can be practically used in the clinic. We further believe that the techniques we have developed to determine the required adaptive phase corrections will be of interest to the wider, very active, community developing adaptive endoscopes.
Sectors Healthcare,Manufacturing, including Industrial Biotechology,Pharmaceuticals and Medical Biotechnology,Other

Description This project provided valuable experience for project research staff who gained interdisciplinary training at the interface between physics and medicine. These staff have now moved on to: a permanent academic position at a UK university, a research fellowship at a UK university, a UK microscopy facility position and a software developer position at a UK SME. Our first demonstration of the use of a negative curvature fibre for delivering femtosecond infrared radiation for non-linear optical microscopy has attracted interest from a number of companies including a UK SME and we have been able to provide them with advice and information on our experience with these fibres. Our first demonstration of an adaptive multiphoton endoscope capable of imaging while the fibre is perturbed and with all measurements performed at the proximal end is an important milestone, but it will take significant further development to turn this into a clinical instrument. We are working to develop new projects utilising these instruments. In particular, we aim to explore the intra-operative application of the handheld multiphoton microscope, e.g. for demarcating tumour margins in brain surgery, as well as continuing with its application to skin cancer. The adaptive endoscope project presented intriguing challenges and opportunities for this technology, which we hope to further develop to realise its translation to in vivo imaging.
First Year Of Impact 2016
Sector Other
Impact Types Economic

Title Minimizing group index variations in a multicore endoscope fiber 
Description Data for results in "Minimizing group index variations in a multicore endoscope fiber" paper. 
Type Of Material Database/Collection of data 
Year Produced 2015 
Provided To Others? Yes  
Description Collaboration with Jonathan Knight at the University of Bath 
Organisation University of Bath
Country United Kingdom 
Sector Academic/University 
PI Contribution Our team is developing novel multiphoton microscope and endoscope systems utilizing novel optical fibres and fibre bundles developed at the University of Bath.
Collaborator Contribution The University of Bath are developing novel optical fibres and optical fibre bundles.
Impact Project is still underway.
Start Year 2013
Title Use of negative curvature fibre for delivering ultrafast infrared laser pulses for multiphoton microscopy 
Description Conventional single mode optical fibres do not allow ultrafast (~100 fs) near infrared laser pulses to be delivered for multiphoton microscopy without experiencing spectral and/or temporal broadening due to nonlinear effects. Photonic band gap fibres allow near infrared laser pulses to be delivered without nonlinear effects at specific wavelengths but do not allow the transmitted wavelength to be tuned. Working with collaborators at the University of Bath, we showed for the first time that negative curvature fibre produced at the University of Bath provides the ability to deliver ultrafast near infrared laser pulses for multiphoton microscopy across a broad spectral range and without the need for precompensation of pulse chirp. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2015 
Impact We developed a hand-held multiphoton imaging system using negative curvature fibre for pulse delivery and demonstrated its capabilities across a range of excitation wavelengths without need to perform any realignment of the system. 
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 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 School visit (Horsted Keynes) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact I presented the impact of biophotonics and imaging technology on the world around us and on medicine and drug discovery
Year(s) Of Engagement Activity 2017
Description Sigtuna workshop, Sweden 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact Conference presentation at international workshop.
Year(s) Of Engagement Activity 2013