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Investigating Fluorescence Lifetime Spectroscopy & Imaging
David Birch
Department of PhysicsUniversity of StrathclydeGlasgow G4 0NGScotland
Content
• Fluorescence lifetime and its measurement
• Use in spectroscopy and microscopy
• Applications –
1. Glucose sensing
2. ββββ-amyloid fibrils
3. Nanoparticle metrology
» Focus on aromatic fluorophores
Fluorescence is the emission of light following absorption of light – has a multidimensional signature
,
intensity
=excitation wavelength
=emission wavelength
polarisation
position
time
Fluorescence ( , , , , )
exc
em
exc em
p
t
I
r
f I p r tλ
λ
λ
λ
=
=
=
=
=
Fluorescence colours
give contrast/specificity
- imaging, microscopy
Fluorescence measurands
,
intensity
=excitation wavelength
=emission wavelength
polarisation
position
time
Fluorescence ( , , , , )
exc
em
exc em
p
t
I
r
f I p r tλ
λ
λ
λ
=
=
=
=
=
⇒ Quantum yield
⇒ Absorption spectrum
⇒ Fluorescence spectrum
⇒ Fluorescence anisotropy
⇒ Fluorescence microscopy
⇒ Fluorescence lifetime decay
Some can be combined
1. Isotropic
2. Emission at longer wavelength than excitation (Stokes shift)
3. Emission spectrum does not vary with excitation wavelength
4. Emission spectrum often a mirror image of the excitation spectrum
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
1
220 240 260 280 300 320 340 360 380 400 420 440 460
Fluorescence spectrum
Iexc(λ)
Ifluor(λ)
I0(λ)
Absorption spectrum
I(λ )
Excited State
GroundState
Abs Fluor
Non-radiative
Relaxation
(Heat)
Wavelength/nm
Intensity
PPONATAHSA
FluorAbs
Fluorescence
Vibrational levels
Electronic level
(Heat)
Fluorescence rate parameters
From steady state excitation From time-resolved excitation
⇒ Fluorescence quantum yield Φ ⇒ Fluorescence lifetime τ
= kr / (kr+knr)
Φ = rate of fluorescencerate of absorption (total decay rate)
τ = average time in exc. state
τ = 1 / total decay rate
= 1 / (kr+knr)
τ = Φ / kr
Φ a difficult, inaccurate
& relative measurement
Range 0-1
τ an easier, accurate & absolute measurement
Typically 10-9 s (ns)
kr radiative rate Knr non-radiative rate
In dilute solution most fl. molecules show a mono-
exponential fluorescence lifetime after ∂ fn excitation
−=
τ
tti exp)(
τ τ τ τ = 2 ns
i(τ)=1/e=0.37i(τ)=1/e=0.37
∂ fn exc.
37% t = 0
intensity at t =τ
τ = 2ns
Real photon counting data has known noise statistics - Poisson
1 std. dev. at each datum
= √Ni
Where Ni is the
number of counts
at channel/time i
Fitting to data
Weighted
residuals
Fitted fn.
∂ fn exc.
Fl. decay
That is the basics – now some refinements….
A. The decay can be complex
Answer: use a sum of n exponentials or other
function
B. We usually don’t have ∂ fn exc.
Answer:
1. Treat the excitation pulse as a
series of ∂ fns
2. The fluor. decay as a series of
exponentials from each ∂ fn
3. Fit the measured data to a
reconstituted decay fn using
statistical criteria to assess
goodness of fit >>
gives “best fit” parameters τ,α ...
Above is reconvolution
I(t) = Σ αi exp(–t/τi )i=1
n
Typical reconvolution fit to data
Exc. ≠ ∂ fn
Decay = Σ ∂ fn responses
Exc. pulse = Σ ∂ fns
Fitted fn.
Residuals
A poor fit – model is incorrect
2 exp. fit to 5 exp. data
High chi-sq
Systematic not random trend
Fit & data differ
0
2000
4000
6000
8000
10000
12000
1 2 3 4 5 6 7 8 9 10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
TIME, CHANNELS
PH
OT
ON
CO
UN
TS
S
Pulsed source(e.g. LED)
stop/start ≤ 2% avoids data pile-up
statisticalsingle photon
events
periodic pulses
Time digitizer
Em. filter
t
Time- correlated single-photon counting
Cumulative
histogram
COMPUTER
DECAY
ANALYSIS
Photomultiplier Detector
Instruments –
Integrated or modular?
Advantages of Fluorescence Lifetimes
Easy, accurate & absolute measurement
Digital not analogue technique - single-photon sensitivity & known statistics
Gives kinetic rates & dynamic information
Concentration independent – overcomes photo-bleaching
Extra specificity – can discriminate against background fluorescence,
scattered excitation etc
Includes fluorescence virtues - Stokes red shift, single-molecule
detection, compatible with optoelectronics (200 – 1000 nm)
Probe of nano-environment – pH, polarity, quenching, energy transfer, analyte specific interactions, etc
1st application – in-vivo glucose sensing
The Problem of Diabetes Mellitus -
• A condition characterized by a chronically raised blood glucose concentration due to a relative or absolute lack of insulin (Type I)
• 250 million people have diabetes worldwide (~ 7.5 x HIV/AIDS)
• 3.8 million people die from diabetes every year
• 10% of healthcare spending in many countries
• Maintaining normal glucose levels minimises complications –blindness, kidney disease, arteriosclerosis, poor circulation
• Grand challenge - No non-invasive and continuous glucose sensor exists
• Glucose metabolic range ~ 5 – 30 mM
Present-day blood glucose monitoring
• Impaired signal responses and drift in serum and tissue due to other metabolite interference
• Requires frequent calibration and impairs accuracy
• Not continuous – problem when sleeping, driving
• Poor patient compliance
We need a new technology
Fluorescence sensing?
– based on finger-prick sampling & electrochemical monitoring
Problems!
Many important metabolites don’t absorb or fluoresce at all in the visible
- glucose is one of them unfortunately!
& melanin absorption & scatter increases towards the UV
Excited State
GroundState
Abs Fluor
Non-radiative
Relaxation
(Heat)
Vibrational levels
Electronic level
One possibility - Multi-photon excitation
2 x 800 nm ≡ 1 x 400 nm
5 x 800 nm ≡ 1 x 160 nm
Photon energies add
Excited State
GroundState
Abs Fluor
Non-radiative
Relaxation
(Heat)
Vibrational levels
Electronic level
Multi-photon excitation
2 x 800 nm ≡ 1 x 400 nm, 5 x 800 nm ≡ 1 x 160 nm
Linear alkane fluor. decay at 220 nm
A Volmer, K Wynne and D Birch, Chem.Phys.Letts. 299, 395,1999
# o
f carb
on
s
time / ns
Photon energies add
But multi-photon excitation needs femtosecond laser exc. -Expensive, complex and impractical for a miniaturized sensor
Also – UV fluorescence will be filtered out by melanin
Answer –
use an extrinsic fluorescent probe & an indirect assay
Two approaches to fluorescence-based
glucose sensors
Fibre-optic glucose sensor in the
subcutaneous tissue
‘Smart tattoo’ concept
of implanted glucose
micro- or nanosensors for
non-invasive monitoring
Excitation Fluorescence
Fluorescence indirect assays
Receptor
Glucose
Change in
fluorescence
Lectin (Con A)
Enzyme (glucose oxidase, hexokinase)
Boronic acid derivative
Cell
Bacterial glucose-binding protein
Promising approach -
Glucose sensing with glucose binding protein(GBP)
Engineer mutant of GBP with cysteine near glucose binding site
Attach environmentally sensitive fluorophore (badan) to the cysteine
Hinge protein closes around
glucose on binding to protein & decreases polarity around dye as water is extruded
Φ & τ of badan increases
Reversible
C terminus
N terminusGlucose
Badan attached
at Cysteine 152
Khan F, Gnudi L, Pickup JC.
Biochem Biophys Res Comm. 2008; 365: 102-106.
Badan fluorescence spectra
- polarity sensitive
1) toluene
2) chloroform
3) acetonitrile
4) ethanol,
5) methanol
6) water.
Badan - 6-bromoacetyl-2-dimethylaminonaphthalene
Φ
Fluorescence lifetime of badan
Fluorescence
decay in water
Fluorescence decay in organic solvent
In Water
Glucose increases encapsulated GBP-Badan fluorescence by ~ 300%
GBP-Badan capsules without glucose
GBP-Badan capsules with
100 µM glucose
λex = 400 nm
λem = 550 nm
GBP-Badan capsules without glucose
GBP-Badan capsules with
100 µM glucose
λex = 400 nm
λem = 550 nm
CaCO3CaCO3 CaCO3
GBP-B
GBP-Badan poly-lysine and poly-
glutamic acid layers
Template
dissolution
with EDTA
5µm
Encapsulation -
GBP-badan fluorescence lifetime increases on addition of glucose
Bi-exponential decay
Glucose changes % of :
Short lifetime component ~0.8 ns
Long-lifetime component ~3.1 ns
310 µM glucose
Zero glucose
Open form GBP-badan
Closed form GBPOn glucose binding
Test responses of GBP-badan
0.1 1 10 100 1000
100
200
Kd ~ 14 mM
PBS
Kd ~ 12 mM Serum
[glucose] mM
∆∆ ∆∆ F
luo
res
ce
nc
e
PBS
Serum
∆τ
⁄%τ
Glucose / mMGlucose / mM
Capsules (tattoo) Agarose beads (fibre optic tip)
In glucose solution
T Saxl, F Khan, M Ferla, D Birch, J Pickup
The Analyst (In press)
T Saxl et al Biosens. Bioelectron.
2009; 24: 3229-34.
Fluorescence Lifetime Imaging Microscopy (FLIM)
Contrast-
Each pixel gives lifetime & intensity
Scanning microscope
Pulsed source
Single detector
Single-photon timing
Optimising Agarose beads with FLIM of bound GBP-Badan in PBS. excitation 400 nm, fluorescence 550 nm
fl. intensity image
Fluorescence lifetime images of beads(1 exp fit)
Zero glucose saturated (100 mM glucose)
Glucose sensing summary
• Reliable and accurate glucose monitoring is a major problem in diabetes awaiting a solution
• Fluorophore-labelled glucose-binding protein encapsulated in nano-engineered capsules or immobilised on a fibre optic probe has potential for in-vivo glucose sensing
• System can be miniaturized on an ASIC using pulsed or phase techniques with LED excitation, photodiode detection, average decay time determination, fibre optic coupling
• Recent review
Nanomedicine and its potential in diabetes research and practice
J C Pickup, Z-L Zhi, F Khan, T Saxl and D J S Birch
Diabetes/Metabolism Res. and Rev. 24, 604-610, 2008.
2nd application - ββββ-amyloid (Aβ) fibrils
Uses amino acid intrinsic fluorescence to monitor early stages
of β-amyloid aggregation
Why study fibrils?
Amyloid fibrils play a key role in neurology
e.g. Alzheimer’s, Parkinson’s, Huntington’s diseases, CJD form β -sheets of amyloid plaques in the brain
Alzheimer’s - Afflicts 5% men and 6% women over 60 > 20 m afflicted, expected to be >100 m by 2050
– no clinical diagnosis, no cure
Also in type II diabetes, cataracts
The growth mechanisms of fibrils are poorly understood
Understanding fibril growth may lead to new materials (as good as some old ones!)
The 3 fluorescent amino acids
Pulsed LEDs are now the appropriate sources for these wavelengths
See
Meas. Sci. Technol. 15, L19-22, 2004 ⇒ 280 nm
Appl. Phys. Letts. 86, 261911, 2005 ⇒ 295 nm
Appl. Phys. Letts 89, 63901, 2006 ⇒ 265 nm
Peak
absorption
The 3 fluorescent amino acids
Pulsed LEDs are now the appropriate sources for these wavelengths
See
Meas. Sci. Technol. 15, L19-22, 2004 ⇒ 280 nm
Appl. Phys. Letts. 86, 261911, 2005 ⇒ 295 nm
Appl. Phys. Letts 89, 63901, 2006 ⇒ 265 nm
Peak
absorption
β-amyloid - a protein fragmentThe peptide
One tyrosine (in position 10)
No tryptophan ∴∴∴∴ no energy transfer
40 - 42 amino acids
~ 10-8 M concentration in biological fluids(role unknown)
Amphipathic nature - aggregation
Early oligomers not ββββ -sheets/plaques are thought to be the most cytotoxic
Plaques – symptom not cause
Forms fibrils
& plaques in the brain
200 220 240 260 280
0
5
10
15
Flu
ore
sce
nce
Inte
nsity /
a.u
.
λ /nm
226
280
Absorption/excitation spectra of Tyr in Aβ.
ββββ-amyloid’s single tyrosine -absorption & fluorescence spectra
Fluorescence spectra of Tyr in Aβ
300 310 320 330 340 350
10
20
30
40
50
60
Tyr emission spectra
in beta-amyloid
Flu
ore
sce
nc
e I
nte
nsit
y
/a
.u.
Wavelenght /nm
Ex@279nm
λ/nm
500 550 600 6500
150
300
450
600
750
Flu
ore
scen
ce I
nte
nsity / a
.u.
λ (nm)
t / h:
341
314
247
219
80
55
29
0.8
Evolution of emission spectra of ThT in time
Thyoflavin T (ThT)– extrinsic probe traditionally used
to detect ββββ-amyloid aggregation
Increasingly planar configuration during
ββββ-amyloid aggregation increases fluorescence
0 60 120 180 240 300 3600
100
200
300
400
500
600
700
800
t / h
Pea
k I
nte
nsity / a
.u.
Evolution of emission intensity of ThT in time at peak intensity (482nm)
No
measurable
change
Problems –
Early stage aggregates are most neurotoxic, but not detected by ThT
ThT needs ββββ-sheets to be formed first
Extrinsic probes can
disturb native structure
Thyoflavin T (ThT) – extrinsic probe traditionally
used to detect ββββ-amyloid aggregationT
hT
0 50 100 150 200 250 300 3500
2
4
6
8
τ3
τ2
τ
/ n
sτ
1
t / h
ββββ-amyloid’s tyrosine fluorescence decay- 3 exponential decay components
- τ1, τ2, τ3 correspond to tyrosine rotamers
ττττ constant
in early
stages ∴∴∴∴ φφφφ constant
componentcontributions to the fluorescence fi
track decayamplitudes & rotamerpopulations pi
i(t) = ΣΣΣΣ αi e –t /ττττi
i=3
fi = αiττττi /ΣΣΣΣi=3 αiττττI
fi ~ φφφφi x pi
0 50 100 150 200 250 300 3500.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
p3
p2
p1p
i
t / h
ββββ-amyloid tyrosine decay parameters.
Populations p1, p2, p3 derived from decay amplitudes α1, α2, α3
Interpreted as aggregation affecting tyrosine rotamer distributions
HN
CH
C
C
NH
O
R
C
O
R
O H
H
H
α
β
Rotamers at
180°,60°, -60°
0 15 30 45 60
0.30
0.35
0.40
0.45
p1 / p
2
t / h
1
2
3
4
Ft /
Fo
Ratio between amplitudes p1and p2 from Tyr
and between Ft and F0 from ThT (pH 7, 30 µM Aβ, 22C)
Comparison between tyrosine and ThT
~ 30% change
Tyr ThT
For further details see
O Rolinski, M Amaro & D Birch Biosens. & Bioelec. 25, 2249, 2010
Negligible change
Tyr
ThT
ββββ-Amyloid fibrils summary
Tyrosine fluorescence lifetime reports on early oligomer
aggregation -
Offers a new approach for research into drug
intervention therapeutics
3rd application - nanoparticle metrology
Concerns over possible nanoparticle toxicology and risk
to the environment
Fear of the unknown – the next asbestos?
Risks unknown
Correlation of effect with size unknown
Size often unknown
3rd application - nanoparticle metrology
Concerns over possible nanoparticle toxicology
Fear of the unknown – the next asbestos?
Risks unknown
Toxicology correlation with size unknown
Size often unknown
1-10 nm particles of particular concern –pass through cell membranes & are the hardest to measure
Conventional Techniques for Np metrology
x-ray, neutron scattering techniques –-works over limited Np concentration range-difficult to use for on-line measurements
-complex
Light scattering - Inadequate resolution < 10 nm
Electron micrographs & AFM
-a direct visualisation, but only on dry samples, not in-situ
Nitrogen isotherms and mercury porosimetry-only on dry samples, slow and expensive
Fluorescence lifetime metrology – ideal for 1 -10 nm
Metrology ?How can fluorescence lifetimes measure distance?
Polarised fluorescence anisotropy decay time τrgives radius r via Stokes-Einstein eq.
Non-radiativeFluorescence resonance energy transfer (FRET) rate k(r) between donor D and acceptor A quenches decay time
- Requires D-A spectral overlap
( )τ
=
6
0
0
1 Rk r
r D
A
δ(0)
I(t)
k(r)
34
3rot
B
r
k T
πητ =r
r
I(t) = αexp-(t/τ0 +k(r))
Measurement geometry Random molecule orientation
Iv(t)
Ih(t)
x
y
z
Polariser
Fluorescence anisotropy
Exc.
Fl.
Iv(t)
Ih(t)
x
y
z
Anisotropy introduced
Abs. probability ~ cos2θ
Iv(t)
Ih(t)
x
y
z
Anisotropy destroyed in ns by Brownian rotation
( )( ) ( )
( ) ( )2
v h
v h
I t I tr t
I t I t
−=
+
Ih(t)
Iv(t)
( ) 0rott
r t r eτ−=
34
3rot
B
r
k T
πητ =
Application to silica nanoparticles- the idea is straight forward
Free dye
τr ~ 200 ps
- Gives microviscosity
Dye bound to SiO2 nanoparticle
τr ~ 200 ns
R = 6 nm
Dye electrostatically (or covalently) attached to silica particles
Stable colloidal silica – Dupont’s Ludox© AM30, pH 8.9
+
++
+
+
+
+
_
_
_ _
___
_
_
_
_
_
η - Gives particle size
Colloidal silica (Ludox© AM30) + fluorophore
A negatively charged fluorophore added to SiO2 with a negative surface charge
Fluorescein
ττττ ~ 4 ns
No binding
Before silica added ~ 200 psAfter silica added ~ 300 ps
--Ve
Colloidal silica (Ludox© AM30) + fluorophoreA positively charged fluorophore added to SiO2
with a negative surface charge
Rhodamine 6G
ττττ ~ 4 ns
Before silica added ~ 300 psAfter silica added ~ inf
+
Strong binding. But ττττ << τrHence no dynamic range
-Ve
Colloidal silica (Ludox© AM30) + fluorophore
A positively charged fluorophore added to SiO2
with a negative surface charge
6-Methoxyquinolinium
ττττ ~ 25 ns ~ ττττr / 10
Before silica added ~ 40 psAfter silica added
+
Strong interaction…. and dynamic range!
-Ve
ττττr1 ~ 17 ns
ττττr2 ~ 273 ns
ττττr3 ~ infinity
r
Ludox ττττr2 / ns rfl /nm r (SEM)
SM30 65 ± 15 4.0 ± 0.4 3.5 nm
AM30 273 ± 60 6.4 ± 0.5 6 nm
AS30 1400 ± 590 11.0 ± 1.6 11 nm
6-MQ attached to different Ludox©
-stable silica colloids
Particle rotationaldiffusion
Good agreement
©
Silica nanoparticle growth in a sol-gel
Na2O.SiO2.H2O + H2SO4 → Si(OH)4 + Na2SO4
Si(OH)4 + Si(OH)4 → 2(SiO2) + 4(H2O) – siloxane bonds formed
Sub nm resolution
pH 10, 2% SiO2, tg 250 hr
Nanoparticle summary
Fluorescence anisotropy decay reliably measures
nanoparticle size ⇒⇒⇒⇒
In situ
At comparatively low cost
With ease of use
And high resolution
See
K Apperson, J Karolin, R W Martin and D J S Birch
Meas. Sci. Technol. 20, 25310, 2009
To Conclude…. Fluorescence Lifetimes
• Routine to measure
• Provide more information than steady-state fluorimetry
e.g. kinetics processes
• Versatile probe of nano-environment
• Combine with spectroscopy, microscopy & sensors
• Providing new approaches in helping to solve global
healthcare problems
Acknowledgements
Strathclyde University:
Olaf Rolinski - FRET & Aβ
Jan Karolin – nanoparticles
Mariana Amaro - Aβ
KCL School of Medicine:
John Pickup - Glucose
& co-workers
Horiba Jobin Yvon IBH Ltd:
David McLoskey - UV LEDs
Kulwinder Sagoo- UV LEDs
Sponsors:
EPSRC
Scottish Funding Council
Wellcome Trust
& thank you for attending this Webinar
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