Development of a single channel hyperspectral fluorescence lifetime instrument

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

Abstract

Fluorescence lifetime imaging (FLIM) provides a powerful optical imaging modality that may be used to contrast different types of fluorescent molecule (called 'fluorophores') or to provide information concerning the local fluorophore environment. While 'conventional' fluorescence intensity imaging is widely used for molecular biology to visualise distributions of proteins that have been 'labelled' by attaching them to convenient fluorophores, it is relatively difficult to obtain quantitative data concerning factors that affect the efficiency of the fluorescence process. This is important because, in principle, the fluorescence efficiency is a function of the local fluorophore environment and can give information concerning what is happening to the fluorophore, whereas conventional fluorescence intensity imaging merely reports where it is located. Intensity-based measurements of fluorescence efficiency can be unreliable because of variations in factors such as attenuation, fluorophore concentration or optical pathlength, which can be very difficult to quantify. Fluorescence lifetime measurements are insensitive to these factors. This makes FLIM useful for the new technique of Forster Resonant Energy transfer (FRET) where fluorescence is quenched (diminished) by adjacent fluorophores. This quenching of fluorescence by energy transfer between molecules requires them to be within ~ 10 nm and so this provides a means for biologists to image when pairs of proteins are interacting. FLIM is useful because the quenched fluorophores exhibit a shorter fluorescence lifetime. At Imperial we have a range of interdisciplinary research programmes exploiting FLIM including FLIM-FRET imaging of inter-cell signalling and signal pathways within cells, which are important to understand the mechanism underlying diseases such as cancer. Successful FRET experiments require careful optimisation of fluorophore labelling that must be tested by control experiments. Other FLIM experiments require characterisation of the radiation emitted by fluorescence probes. Our current tool of choice for FLIM & FRET is a confocal microscope but, with many biologists and other collaborators competing for time on this expensive instrument, we have a major bottleneck that is limiting research progress. The proposed new tool would permit FRET and control experiments on protein or fluorophores to be done 'off-line'. It would also be useful to characterise fluorophores for many other FLIM experiments. Furthermore it would be user-friendly and relatively easy to replicate so we could envisage spreading this technique to our collaborators within Imperial's life science departments and at the Institute of Cancer Research (ICR). This would have a very positive impact on progress towards establishing new FLIM and FRET experiments and would also greatly relieve the congestion on our confocal microscope system. We measure fluorescence lifetime by exciting molecules with a short pulse of light and observing how they lose this energy by emitting photons. The wavelength of the photons emitted and the timescale over the fluorescence signal decays are characteristic of the different fluorophore molecules. For this project we aim to develop an automatic hyperspectral fluorescence lifetime measurement system that would simultaneously record the fluorescence emission in terms of the wavelength and temporal decay (lifetime) profiles to provide a full spectro-temporal characterisation of the fluorophore emission. This new tool will be applicable to cuvette measurements and homogenous assays in multiwell plate arrays, which in high-throughput screening are used for drug discovery. It will also be configurable with fibre-optic probes for in situ point measurements.

Technical Summary

We aim to develop a single-channel hyperspectral fluorescence lifetime imaging (FLIM) system combined with an electronically tunable excitation source to permit full excitation-emission-lifetime matrices to be routinely recorded. This new tool will be applicable to cuvette measurements and homogenous assays in well plate arrays. It will also be configurable with fibre-optic probes for in situ point measurements in diverse samples including bioreactors and tissue. Our particular applications relate to FLIM microscopy including FLIM-FRET imaging of phosphorylation at the immune synapse of NK cells and GTP-ras binding. Successful FRET experiments require careful optimisation of probe expression and control experiments. Currently we use a confocal microscope with time-correlated single photon counting (TCSPC) for FRET but with up to 10 users competing for time on this instrument, we have a major bottleneck that is limiting research progress. The proposed instrument would facilitate non-imaging FRET and control experiments on homogenous protein or other solutions and spatially integrated measurements of cells. Other FLIM projects also require temporal and spectral characterisation of fluorescence probes for which this would provide a valuable resource - particularly to validate our novel wide-field hyperspectral FLIM microscopes. Because this new instrument would be user-friendly and relatively easy to replicate, we could envisage spreading this technique to our life science collaborators within Imperial and at the ICR. This would positive impact progress towards establishing new FLIM and FRET experiments and would also greatly relieve the congestion on our confocal microscope system. We have already demonstrated the efficacy of an electronically tunable source for FLIM and here propose to use a new commercially available all-fibre integrated system that we will adapt for electronic tunability.

Publications

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Description Fluorescence lifetime imaging (FLIM) provides a powerful optical imaging modality that may be used to contrast different types of fluorescent molecule (called "fluorophores") or to provide information concerning the local fluorophore environment. While "conventional" fluorescence intensity imaging is widely used for molecular biology to visualise distributions of proteins that have been "labelled" by attaching them to convenient fluorophores, it is relatively difficult to obtain quantitative data concerning factors that affect the efficiency of the fluorescence process. This is important because the fluorescence efficiency is a function of the local fluorophore environment and can give information concerning what is happening to a fluorophore, whereas conventional fluorescence intensity imaging merely reports where it is located. Intensity-based measurements of fluorescence efficiency can be unreliable because of unknown variations in factors such as attenuation, fluorophore concentration or optical path length but fluorescence lifetime measurements are insensitive to these factors. This makes them useful for studying the fluorescence properties of biological tissue and for quantitative measurements in cell biology. A key application is the technique of Forster Resonant Energy transfer (FRET) where fluorescence is quenched (diminished) by adjacent fluorophores. Because quenching of fluorescence by FRET between molecules requires them to be within ~ 10 nm and results in a shorter fluorescence lifetime, FLIM can provide a robust means for biologists to map out where proteins are interacting, e.g. in cells. At Imperial we have a range of interdisciplinary research programmes exploiting FLIM, often in combination with spectral and polarisation-resolved imaging, including FLIM-FRET imaging of inter-cell signalling and signal pathways within cells, which are important to help understand diseases such as cancer.
The main output of this grant was the development and evaluation of a novel multidimensional fluorimeter exploiting a tunable supercontinuum-based laser source and resolving excitation and emission spectra, as well as lifetime and polarisation in a single instrument - and in a single measurement if required. This instrument is enclosed on a breadboard of 90x60 cm and so is portable as well as convenient. It is designed to be used with cuvettes as with conventional fluorimeters but also incorporates a fibre-optic probe to permit studies of remote samples in situ, such as biological tissue and in culture media, or in vivo, and which can be readily applied to multiwell plate readers. It is fully computer controlled and so permits rapid analysis of multiple samples. The internal supercontinuum source provides excitation from ~ 400 to >800 nm and the instrument has been designed to permit rapid coupling of external excitation sources as required, e.g. u.v. lasers for exciting endogenous fluorescence.
This combination of all these measurement modalities in a compact "hands-off" instrument exploiting recently available fibre laser-based supercontinuum technology to provide tunable excitation is unique and highly enabling. Within the project it has been validated with fluorescence standards and has been applied to FRET measurements in solution, to characterise a new fluorescence-based membrane probe, to study staurosporine and to provide a label-free readout of cartilage degeneration in fresh tissue.
Exploitation Route This new instrument has a vast range of applications, many of which we have subsequently demonstrated including in vivo label-free needle biopsy of patients or ex vivo tissue, solution-based FRET studies of molecular interactions, readouts of metabolic changes in humans, animals and plants, and characterisation of fluorescence probes. It is a convenient way to access a wide range of data from fluorescent samples.
Sectors Agriculture, Food and Drink,Chemicals,Energy,Environment,Healthcare,Pharmaceuticals and Medical Biotechnology,Security and Diplomacy

 
Description This project provided an versatile time-resolved spectrophotometer that formed the basis for a series of instruments that we have developed and applied to the study of disease (cancer, heart disease and osteoarthritis), including clinical trials of tissue autofluorescence measurements supported by subsequent EPSRC and BHF funding, and most recently adapted to study metabolic changes in plants via time-resolved measurements of chlorophyll autofluorescence to readout the impact of the application of agrochemicals.
First Year Of Impact 2008
Sector Agriculture, Food and Drink,Education,Healthcare
 
Description BBSRC Responsive mode BB/H00713X/1
Amount £473,776 (GBP)
Funding ID BB/H00713X/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2010 
End 03/2013
 
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 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 Hyperspectral time-resolved fluorometer 
Description The main output of this project was a novel instrument to analyse fluorescence emission with respect to excitation wavelength, emission wavelength, fluorescence decay time and polarisation. This it can be used to obtain multidimensional fluorescence datasets that provide exquisite contrast between different fluorophores or states of fluorophores. Unlike a microscope, all the emitted photons are collected as a single point measurement (i.e. a homogeneous assay) and the resulting large number of photons is sufficient to enable the analysis of complex decay profiles with spectral and polarisation resolution. This can be applied to acquire excitation-emission-lifetime matrices (or subsets thereof), which can be used to study changes in fluorescence to report on the local molecular environment or it can be used to distinguish different fluorophores - for example using the time resolution to discriminate between fluorophores with similar spectral properties. It can also be used for FRET studies. The capability for time-resolved and polarisation-resolved fluorescence measurements enable time-resolved anisotropy studies, e.g. of molecular rotational dynamics. The instrument is designed for solution-based studies in cuvettes and also can make measurements of remote samples via a fibre-optic probe. After this project finished, it was also modified to enable automated homogenous measurements of samples arrayed in multiwell plates. make measurements of remote samples via a fibre-optic probe. After this project finished, it was also modified to enable automated homogenous measurements of samples arrayed in multiwell plates. The internal supercontinuum source provides excitation from ~ 400 to >800 nm and the instrument has been designed to permit rapid coupling of external excitation sources as required, e.g. u.v. lasers for exciting endogenous fluorescence. This combination of all these measurement modalities in a compact "hands-off" instrument exploiting recently available fibre laser-based supercontinuum technology to provide tunable excitation is unique and highly enabling. 
Type Of Technology New/Improved Technique/Technology 
Year Produced 2008 
Impact We have applied this new tool across a broad range of projects and it is still in use today. We use it for reference measurements of fluorophores that are then used to label cells for FLIM studies and we have undertaken solution-based experiments to study molecular confirmations, e.g. to study cell signalling pathways. We have also exploited its capability to make measurements via a fibre-optic probe to in situ point studies of biological tissue. This has been developed in later projects as a tool for clinical studies of cancer, heart disease and osteoarthritis and to clinical diagnosis, including in vivo. 
 
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. 
 
Description Crick retreat 2011 MDFI across scales 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach National
Primary Audience Other audiences
Results and Impact Invited talk, Francis Crick Institute retreat, Ashridge, 2011

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