Transcript DEVELOPING FLUORESCENCE LIFETIME IMAGING ENDOSCOPES …

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
2Cancer
3North
Group, Department of Physics , Imperial College London
Research UK, London Research Institute, United Kingdom
West London Hospitals NHS Trust, United Kingdom
Overview
Section.1 Wide-field FLIM flexible endoscopy
– Wide-field FLIM of autofluorescence for clinical applications
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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
Section.1 Introduction to wide-field FLIM for clinical applications
Basics of FLIM
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E1
Fluorescence reports on the molecules and their
local chemical environment
G k
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
E0
Intensity
~ f{h}, h =
G/(G+k)
Wavelength, l ~ hc/(E1-E0)
Problems: [fluorophore], heterogeneity, scattering and background fluorescence
l
Difficult to make absolute
intensity measurements
Solution: ratiometric measurements  lifetime = 1/(G+K)
Assign fluorescence lifetimes to image pixels & map lifetimes values to a
color space to generate FLIM maps.
Section.1 Introduction To Wide-field FLIM For Clinical Applications
Origins Of Tissue Autofluorescence
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Typical tissue fluorophore are efficiently excited by ultra-violet & visible light to generate
Stokes-shifted visible fluorescence.
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As diseases develop the fluorescence signal from tissue may change as fluorophor
composition & local microenvironment change.
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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 applications
FLIM endoscopy of autofluorescence and a brief look at past research
Conventional white light endoscopy lacks functional information that could be valuable for
clinical procedures
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Fluorescence lifetime imaging provides functional information
Fluorescence lifetime imaging of autofluorescence provides label free contrast
Numerous ex vivo/in vivo point probe studies have demonstrated that fluorescence lifetime
reports on tissue state (Marcu. BMES 2012)
Only two studies of FLIM endoscopy in vivo to date (Mizeret et al. J of fluorescence (1999),
Sun et al. Cambridge University Press(2013))
Examples of published work showing potential clinical value of FLIM
McGinty et al, Biomed. Opt. Expr. 2010
Thomas et al, Phot Chem & Phot Bio. 2010
Galletly et al, B J Dermatol. 2008
Section.1 Introduction To Wide-field FLIM For Clinical Applications
intensity
Wide-field Time-domain FLIM Using Time-gated Detection
Ultrafast laser system
Excitation
pulse Fluorescence
emission
I = I0 e-t/t
t
Delay
generator
CCD
t 1 t2 t 3
Dichroic
mirror
GOI
time
t3
Filter
t
t2
Sample
t
t1
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 Autofluorescence
Macroscopic Imaging Of Ex Vivo Head & Neck Tissue Samples
Ultra-violet & blue MHz Portable system for
repetition rate picoseconds
transporting
pulsed lasers
system to hospitals
Imaging platform
ICCDs & color
camera
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
Section.1 Wide-field FLIM Endoscopy For Tissue Autofluorescence
Characterising System Performance & First Results
Resolution & FOV
Lifetime accuracy demonstration
FLIM measurements of a homogenous fluorescent sample (plastic
sheet)
Fluorescence USAF test chart defines a
macroscopic field of view with sub
millimetre resolution
Fluorescence
intensity
FLIM map Intensity weighted
FLIM map
Demonstration of system with human tissue  FLIM of a laryngectamy
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Fluorescence
intensity
Intensity weighted FLIM
map
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
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
Section.1 Wide-field FLIM Endoscopy For Tissue Autofluorescence
FLIM Of A Laryngeal Sample With A Low Power Compact Blue Diode
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3
A
Acquisition parameters
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Color photo 1 is an in vivo image of a
human vocal cord presenting a tumour
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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.
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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 .
D
B
C
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
Section.1 Wide-field FLIM Endoscopy For Tissue Autofluorescence
Results
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/𝑐𝑚2)
Typical
acquisition
times (s)
355 – 445
12
1
1-4
< 260
< 60
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:
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2.
3.
4.
tissue is not imaged in situ
fluids on the surface may modulate signals i.e. blood
trauma to samples during surgery may modulate signals.
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 Autofluorescence
A Prototype FLIM Endoscope Targeting Autofluorescence
Designing a flexible endoscope that’s compatible with FLIM of tissue autofluorescence
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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
PC for
interfacing
with
hardware
Color
camera
Imaging
Lenses
Flexible
endoscope
Flexible
endoscope
distal optics
in ferrule
Time-gated wide field
FLIM
Time gated ICCD
with picoseconds
resolution
Trigger delay unit for
synchronising
laser with ICCD
Flip
mirror
MHz Pulsed
MHz
Pulsed Laser
Laser
for
UV
– Blue
for UV – Blue
excitation
excitation
Multimode
fibre for
illumination
Excitation light coupling lens with NA
matching fibre NA
prototype endoscope integrated with a wide-field time-gated
FLIM detector & pulsed light source
Section.1 Wide-field FLIM Endoscopy For Tissue Autofluorescence
A Prototype FLIM Endoscope Targeting Autofluorescence
Characterising resolution & lifetime accuracy of FLIM through prototype endoscope
Steel ferrule to house optics
Illumination spot does
not overlap completely
with field of view
Endoscope distal optics
Imaging fluorescent
USAF chart indicates
smallest resolvable
features are ~30 µm
~3 mm
FOV
Coherent fibre bundle
Protective jacket
GRIN lens
Multimode excitation fibre ~4 mm working
distance
Lifetime accuracy demonstration by imaging homogenous sample of
Coumarin-6 reference dye dissolved in ethanol when using 445 nm excitation
Fluorescence
intensity (a.u)
FLIM map
(picoseconds)
Lifetime distribution of FLIM map
(picoseconds)
Section.1 Wide-field FLIM Endoscopy For Tissue Autofluorescence
A 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
Intensity weighted
colour camera photos FLIM images
Acquisition parameters
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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 Autofluorescence
Summary
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 Applications
FRET efficiency E
Basics Of Forster Resonance Energy Transfer (FRET)
1
0.8
E
0.6
0.4
0.2
1
,
6
1  R R0 
R0 ~ nm
0
0
1
2
R/R0
E  1
 DA
D
Section.2 Introduction To Confocal FLIM For Biomedical Applications
Basics Of Forster Resonance Energy Transfer (FRET)
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.
Example diagram of an
intramolecular FRET single
chain biosensor
FLIM
endoscope
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 FRET
A CLSE FLIM Endoscope
Optically-sectioned subcellular resolution FLIM endoscopy system
 Commercial 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 plane
 The 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
FRET constructs of
varying linker lengths
FLIM map
Intensity weighted Fluorescence
FLIM map
intensity
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V . Koushik et al Biophysical J. 2006
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.
Section.2 Developing CLSE FLIM endoscopy for FLIM FRET
Summary
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CLSE adapted for optically sectioned FLIM FRET with subcellular resolution
Demonstration of potential to read out protein interactions by FRET using FRET
standards
Future outlook
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Apply instrument to in vivo imaging of FRET sensors to investigate value of method
to biomedical research
“Thank you”
Kentech Instruments