DEVELOPING FLUORESCENCE LIFETIME IMAGING ENDOSCOPES FOR BIOMEDICAL APPLICATIONS

Hugh Sparks1, Ian Munro1, Douglas Kelly1, Sean Warren1, Gordon Kennedy1, Eishu Hirata2, Esra Nigar3, Eric Sahai2, Taran Tatla3, Christopher Dunsby1 and Paul French1

1Photonics Group, Department of Physics , Imperial College London

2Cancer Research UK, London Research Institute, United Kingdom

3North West London Hospitals NHS Trust, United Kingdom

OverviewSection.1 Wide-field FLIM flexible endoscopy

– Wide-field FLIM of autofluorescence for clinical applications• Basics of FLIM

• Origins of tissue autofluorescence

• FLIM endoscopy of autofluorescence and a brief look at past research

• Time-domain FLIM using time-gated detection

– Wide-field FLIM endoscopy of tissue autofluorescence • Macroscopic imaging of ex vivo head & neck tissue samples

• A prototype FLIM endoscope targeting autofluorescence

• Summary of work and future outlook

Section.2 Confocal laser scanning endoscope (CLSE) adapted for FLIM

– Introduction to confocal FLIM for biomedical applications• Basics of Time Correlated Single Photon Counting (TCSPC)

• Basics of Forster Resonance Energy Transfer (FRET)

– Developing CLSE FLIM for mentoring protein interactions by FLIM FRET• A commercial CLSE system adapted for TCSPC FLIM

• Demonstration of CLSE FLIM FRET in vitro

Intensity

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• Fluorescence reports on the molecules and their local chemical environment

• When combined with imaging it enables the correlation of structure and function - molecular imaging.

• Can target exogenous and/or endogenous fluorescence for functional imaging of biology

Solution: ratiometric measurements lifetime = 1/(+K)Assign fluorescence lifetimes to image pixels & map lifetimes values to acolor space to generate FLIM maps.

Problems: [fluorophore], heterogeneity, scattering and background fluorescence

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Difficult to make absolute intensity measurements

Section.1 Introduction to wide-field FLIM for clinical applicationsBasics of FLIM

• Typical tissue fluorophore are efficiently excited by ultra-violet & visible light to generate Stokes-shifted visible fluorescence.

• As diseases develop the fluorescence signal from tissue may change as fluorophor composition & local microenvironment change.

• Changes in chromophor content (e.g. blood supply) and local architecture (e.g. inflammation) can modulate excitation and emission spectra (Wagnieres et al , Photochem).

Section.1 Introduction To Wide-field FLIM For Clinical ApplicationsOrigins Of Tissue Autofluorescence

Section.1 Introduction to wide-field FLIM for clinical applicationsFLIM endoscopy of autofluorescence and a brief look at past research

McGinty et al, Biomed. Opt. Expr. 2010

Galletly et al, B J Dermatol. 2008

Thomas et al, Phot Chem & Phot Bio. 2010

Examples of published work showing potential clinical value of FLIM

Section.1 Introduction To Wide-field FLIM For Clinical ApplicationsWide-field Time-domain FLIM Using Time-gated Detection

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emissionUltrafast laser system

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Time-gated detectors using GOIs (e.g. Kentech Ltd.) coupled to CCDs (i.e. intensified CCD, ‘ICCD’) provide parallel pixel acquisition of fluorescence lifetime measurements across a CCD field of view. As a result ICCDs can acquire a given lifetime precision faster than photon counting methods such as TCSPC but are less photon efficient (Talbot et al. J of Biophotonics (2008)). For biomedical applications that require wide-field macroscopic images in real-time this method is the preferred choice (Munro et al. J of Biomedical Optics 2005).

Section.1 Wide-field FLIM Endoscopy For Tissue AutofluorescenceMacroscopic Imaging Of Ex Vivo Head & Neck Tissue Samples

Imaging platform ICCDs & color

camera

Portable system for transporting

system to hospitals

Ultra-violet & blue MHz repetition rate picoseconds

pulsed lasers

A portable wide-field FLIM system based on time-gated ICCDs was constructed & taken to Northwick Park Hospital, Ear, Nose & Throat Department, London, UK (NPH).

Key components of system:•Pulsed UV & Blue lasers excite the sample’s fluorophore•Multimode fibre delivers light to sample plane from lasers•Camera lenses image macroscopic fields of view onto ICCDs for FLIM•Color camera beside ICCDS record color photos of samples

Fluorescence USAF test chart defines a macroscopic field of view with sub

millimetre resolution

Resolution & FOV

FLIM measurements of a homogenous fluorescent sample (plastic sheet)

Fluorescence intensity

FLIM map Intensity weighted FLIM map

Lifetime accuracy demonstration

Acquisition parameters•Sample imaged within minutes of resection•2 mW power @ 355 nm •long pass emission filter with a 365 nm cut-on wavelength for collecting fluorescence & rejects excitation •Mono-exponential decays were fitted in each image pixel•3 FLIM images were stitched together to make a larger field of view using ImageJ

Demonstration of system with human tissue FLIM of a laryngectamy

Fluorescence intensity

Intensity weighted FLIM map

Color photo

Section.1 Wide-field FLIM Endoscopy For Tissue AutofluorescenceCharacterising System Performance & First Results

• These initial results demonstrates that system has adequate sensitivity.

• Need further samples and correlative histology to study origin and value of contrast seen.

• Color photo 1 is an in vivo image of a human vocal cord presenting a tumour

• Photos 2 & .3 show the sample ex vivo under white light or using the FLIM system with a blue laser diode for excitation @ 445 nm centre wavelength.

• In Photo.3, lifetimes appear distinct for region A,B,C & D relative to surrounding areas. Specifically in photo 3: region (A) is suspected cancer , (B) suspected carcinoma in situ, (A) everted mucosa and (D) stiches .

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3 3

A B C

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Section.1 Wide-field FLIM Endoscopy For Tissue AutofluorescenceFLIM Of A Laryngeal Sample With A Low Power Compact Blue Diode

Acquisition parameters

•Sample imaged within minutes of resection•1.5 mW power @ 445 nm •Pair of long pass emission filters with a 510 & 475 nm cut-on wavelength collects fluorescence & rejects excitation light•1 ns gates were shifted across a 25 ns window in 1 ns increments•Mono-exponential decays were fitted in each image pixel

Ultra-violet & blue excitation light generated FLIM images of ex vivo human diseased tissue with lifetime distributions typically varying from 1 to 4 nanoseconds

For modest sample plane intensities of ~ 2 mW (compared to commercial light sources used in endoscopic imaging procedures) acquisition times were less than 60 seconds

Origins of tissue contrast are not clear. Measured lifetimes may correlate with disease stage but there are a number of confounding influences on measured values. In particular:

1.tissue is not imaged in situ2.fluids on the surface may modulate signals i.e. blood3.trauma to samples during surgery may modulate signals. 4.lack of normal tissue makes if difficult to correlate lifetime contrast with disease stage.

In order to better understand the clinical value, in vivo measurements are preferable. To this end, custom made flexible endoscopes are being designed which can fit down the working channel of commercial endoscopes…

Section.1 Wide-field FLIM Endoscopy For Tissue AutofluorescenceResults

Excitation wavelength range used (nm)

Number of patients sampled

Typical powers at the sample plane (mW)

Typical lifetime range (ns)

Laser Intensity at sample plane (μW/ 2𝑐𝑚 )

Typical acquisition times (s)

355 – 445 12 1 1 - 4 < 260 < 60

Section.1 Wide-field FLIM Endoscopy For Tissue Autofluorescence A Prototype FLIM Endoscope Targeting Autofluorescence

Designing a flexible endoscope that’s compatible with FLIM of tissue autofluorescence A separate illumination fibre to minimise background noise from autofluorescence of optics. A multimode fibre for illumination with a large NA in order to fill the imaged field of view Image through a 30,000 core flexible coherent bundle with a GRIN lens epoxied to the distal end for imaging

macroscopic fields of view. A protective steel ferrule aligns the excitation fibre and imaging bundle to overlap FOV & illumination Relays image from bundle to a wide-field FLIM detector or white light camera for multimodal imaging Fits down the working channel of commercial endoscopes, i.e. less than 3 mm diameter

prototype endoscope integrated with a wide-field time-gated FLIM detector & pulsed light source

Colorcamera

Time gated ICCD with picosecondsresolution

Trigger delay unit for synchronisinglaser with ICCD

PC for interfacing with hardware

MHz Pulsed Laserfor UV – Blue excitation

MHz Pulsed Laserfor UV – Blue excitation

Flexible endoscope

Multimode fibre for illumination

Imaging Lenses

Excitation light coupling lens with NA matching fibre NA

Time-gated wide fieldFLIM

Flexible endoscope distal optics in ferrule

Flip mirror

Section.1 Wide-field FLIM Endoscopy For Tissue Autofluorescence A Prototype FLIM Endoscope Targeting Autofluorescence

Characterising resolution & lifetime accuracy of FLIM through prototype endoscope

Endoscope distal optics

~4 mm working distance

~3 mm FOV

Steel ferrule to house optics

GRIN lensCoherent fibre bundleProtective jacket

Multimode excitation fibre

Fluorescence intensity (a.u)

FLIM map (picoseconds)

Lifetime distribution of FLIM map(picoseconds)

Imaging fluorescent USAF chart indicates smallest resolvable features are ~30 µm

Lifetime accuracy demonstration by imaging homogenous sample of Coumarin-6 reference dye dissolved in ethanol when using 445 nm excitation

Illumination spot does not overlap completely

with field of view

Section.1 Wide-field FLIM Endoscopy For Tissue AutofluorescenceA Prototype FLIM Endoscope Targeting Autofluorescence

Taking prototype endoscope to NPH & imaging fresh Ex Vivo tissue samples

FLIM endoscopy of human diseased laryngeal tissue (3 samples, 1 FOV for each)

White light colour camera photos

Intensity weighted FLIM images

Acquisition parameters

•Samples imaged with minutes of resection•0.5 mW power @ 445 nm •A pair of long pass emission filters with a 510 & 475 nm cut-on wavelength collected fluorescence•1 ns gates were shifted across a 25 ns window in 1 ns increments•Mono-exponential decays were fitted in each image pixel•All sample images took less than 60 seconds to acquire

Section.1 Wide-field FLIM Endoscopy For Tissue AutofluorescenceSummary

Demonstrated wide-field time-gated FLIM through a flexible endoscope that can fit down the working channel of commercial endoscopes

Demonstrated FLIM of human diseased laryngeal tissue under blue light (445 nm) in less than 60 seconds using a compact laser diode

Blue light is less phototoxic compared to ultra-violet light.

Future outlook

Investigate whether FLIM contrast correlates with tissue state by imaging animal models of cancer.

Investigate optimum excitation light wavelength for clinical applications

Investigate whether in vivo FLIM contrast correlates with tissue state in humans

Reduce acquisition time by increasing laser powers and implementing rapid lifetime acquisition strategies

Section.2 Confocal Laser Scanning Endoscope (CLSE) Adapted For FLIM

Basics Of Confocal TCSPC

Confocal TCSPC

When combined with confocal laser scanning microscopy for time-domain FLIM, TCSPC can be used to generate histograms of photon arrival times relative to laser excitation pulses. Line and frame scanning clocks from the laser beam scanning mechanism assign events to image pixels. Compared to wide-field time-gated FLIM, TCSPC CLSE is more photon efficient but takes more time to achieve a given lifetime precision (Talbot et al. J of Biophotonics (2008)). For quantitative read-outs of protein interactions by FLIM FRET, the sectioning capability of confocal imaging combined with TCSPC can be used to effectively isolate FRET signals from a single image plane with sub-cellular resolution.

Section.2 Introduction To Confocal FLIM For Biomedical ApplicationsBasics Of Forster Resonance Energy Transfer (FRET)

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While in vitro experiments are invaluable to biologists they typically do not reproduce in vivo conditions. In particular, the questions surrounding the exact nature of the biomolecular mechanisms of cancer progression and the efficacy of anti-cancer drugs cannot be fully answered by in vitro experiments. Animals can be used as models of human cancer to more accurately reproduce the biological conditions that influence human cancer. We propose flexible endoscopes integrated with confocal TCSPC FLIM for minimally invasive imaging of FRET biosensors in vivo to allow longitudinal studies of biomolecular processes in animal models. Longitudinal studies should improve the quality of findings and minimise the number of animals needed for a particular study.

Section.2 Introduction To Confocal FLIM For Biomedical ApplicationsBasics Of Forster Resonance Energy Transfer (FRET)

FLIMendoscope

Example diagram of an intramolecular FRET single chain biosensor

The biosensor responds the presence of a substrate causing an increase in FRET efficiency which can be measured by FLIM

FLIM endoscopy can be combined with animal models expressing FRET sensors for minimally invasive functional imaging

Section.2 Developing CLSE FLIM endoscopy for FLIM FRETA CLSE FLIM Endoscope

Optically-sectioned subcellular resolution FLIM endoscopy systemCommercial laser scanning confocal endomicroscope (CLSE),(Mauna Kea technologies, Cellvizio®) adapted for TCSPC. Frequency-doubled tunable (355 - 495 nm) Tai-Sapphire laser (Spectra-Physics, BB Mai Tai). coupled a Cellvizio® scanning unit via a single-mode optical fibre (acting as a pinhole).Dichroic beam splitter transmits fluorescence to the photomultiplier for TCSPC (Becker & Hickl, SPC-830). TCSPC assigns photons to arrival times relative to laser pulses times at the sample planeThe endoscopic probe comprises a coherent fibre optic imaging bundle with a miniature objective at the distal end that provides a 60 µm working distance & 250 µm field of view. Line & frame clocks from the scanning unit register photons to pixels. FLIM data is acquired in FIFO mode to generate “preview” FLIM images in real time based on mean arrival time. Post acquisition processing permits more detailed non-linear fitting analysis.

Section.2 Developing CLSE FLIM Endoscopy For FLIM FRET Demonstration FLIM FRET In Vitro

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FLIM CLSE was applied to image fixed MCF7 cells expressing Cerulean-Venus FRET constructs that present varying degrees of FRET via changes in the length of the linker between the FRET pair. As the linker length decreases, the Cerulean lifetime decreases as expected due to the increase in FRET efficiency.

FRET constructs of varying linker lengths

Fluorescence intensity

FLIM map Intensity weighted FLIM map

V . Koushik et al Biophysical J. 2006

• CLSE adapted for optically sectioned FLIM FRET with subcellular resolution • Demonstration of potential to read out protein interactions by FRET using FRET

standardsFuture outlook

• Apply instrument to in vivo imaging of FRET sensors to investigate value of method to biomedical research

Section.2 Developing CLSE FLIM endoscopy for FLIM FRETSummary

“Thank you”Kentech Instruments