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« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Gaël LatourLaboratoire d’Imagerie et de Modélisation en Neurobiologie et Cancérologie,
Univ. Paris Sud (Orsay)
Microscopie non-linéaire résolue en polarisation pour
l’imagerie structurelle des tissus
multiphoton imaging
of the stroma
(forward and
backward SHG)
Laboratoire d’Optique et Biosciences, Ecole Polytechnique (Palaiseau)
Equipe SHG : I. Gusachenko, M.-C. Schanne-Klein
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Content
�Multiphoton microscopy� principle and setup
� Polarization-resolved SHG� principle
� linear artefacts in P-SHG
� Application to the tendon during
biomechanical assays
� Application to the cornea
� Conclusion and perspectives
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Content
�Multiphoton microscopy� principle and setup
� Polarization-resolved SHG� principle
� linear artefacts in P-SHG
� Application to the tendon during
biomechanical assays
� Application to the cornea
� Conclusion and perspectives
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Endogenous nonlinear signals: the corneaHistological section
(human cornea)
Electron microscopy (TEM)Organization of the stroma
Multiphoton imaging of the cornea
Second harmonic generation (SHG)� stromal collagen fibrils
Two-photon excited fluorescence (2PEF)� epithelial and endothelial cells, keratocytes
� 3D, without staining and multimodal (2PEF, SHG) cornea imaging
Aptel et al., IOVS 51, 2459 (2010)Aptel et al., IOVS 51, 2459 (2010)
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Harmonic generation (SHG)
Induced polarization ...+++= EEEEEEprrrrrrr γβα
linear response non-linear responsestrong excitation necessary
Electric field (2ω) Intensity
I
I x 4
0
SHG �
non-centrosymmetric and dense
distribution of « harmonophores »
� Response @ molecular level
� peptide bond = main harmonophore in biological tissues
� Response @ macro-molecular level
non centrosymmetric medium � β ≠ 0
Electric field (ω)
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Physics of collagen: SHG
Strong SHG response from fibrillar collagens
� Tight alignment of peptide bonds
� Efficient coherent amplification
� At all levels of collagen hierarchical structure:
single helix � triple helix � fibril � fiber
Triple-helix domains[(Gly-X-Y)n]3
Collagen I , II, III, V, XI, …
Form fibrils
Fibrils
Fibers
Form networks
Macromolecular organization
SHG signals from arteries, lung, skin, cornea…Pena et al, J. Am. Chem. Soc. 127, 10314 (2005)
Strupler et al, Opt. Express 15, 4054 (2007)
Deniset-Besseau et al, J. Phys. Chem. B 113, 13437 (2009)
Pena et al, J. Am. Chem. Soc. 127, 10314 (2005)
Strupler et al, Opt. Express 15, 4054 (2007)
Deniset-Besseau et al, J. Phys. Chem. B 113, 13437 (2009)
Molecules
Collagen IV …
SHG silentSHG
Collagen
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Multiphoton microscopy: setup
Excitation
400 600 800 nm
2PEF
SHG
Laser excitation
@730-860 nm
Stack of 2D images vs. z
3D
reconstruction
2PEF SHG
Acquisition
@860 nm
� 20x (0.95 NA) � 0.4 µm x 1.6 µm
� 60x (1.2 NA) � 0.3 µm × 0.9 µm
Resolution
Acquisition time:1-3 s / image (512 x 512 pixels)
z
Multiphoton microscope @LOB
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Content
�Multiphoton microscopy� principle and setup
� Polarization-resolved SHG� principle
� linear artefacts in P-SHG
� Application to the tendon during
biomechanical assays
� Application to the cornea
� Conclusion and perspectives
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Hypotheses:
• С∞v symmetry
• Kleinman symmetry
2 independent tensorial components:
SHG formalism:
SHG tensorial response
SHG Ix
2=ρ
α
SHG Iy
2=ρ
α
� fibril mean orientation (α)
� fibril orientation dispersion in the focal volume (ρ)
Polarimetric SHG ���� quantitative parameters with sub-µm sensitivity
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Kastelic et al., Conn. Tiss. Res. 6, 11-23, 1978
Tendon hierarchical structure
� ≈ 200 µm diameter, 10 cm long tendon fascicle
� surface labeling with fluorescent beads
Rat-tail tendon = model (uniaxial) tissue
The tendon
SHG: collagen fibrils
2PEF: fluorescent micro-beads
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
2PEF
XY scanning
ZEpi-SHG
Filters
Dichroic
mirrors
Objective
(high NA)
Condenser
λ/4
λ/2
Trans-SHG/Y
Trans-SHG/X
Filters
Polarizing
beam-splitter
Analyzer
Ti-Sa
laser
Y
X
Power control
Polarization-resolved SHG: experimental setup
SHG imaging of
rat-tail tendons
200
400
600
0
30
6090
120
150
180
210
240270
300
330
200
400
600
Inte
nsité
SH
G (
a.u.
)
Polarimetric diagram
Incident beam:
�circular or linear polarization
�orientation of the linear polarization
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
z = 39 µm
α = π/4
X Y
50 µmY
X
z = 57 µm
X Y
-π/2 -π/4 π/4 π/20
α [rad]
0 100 200 300100
80
60
40
20
0
SHG signal [arb. units]
z0
De
pth
[μ
m]
100
80
60
40
20
0
De
pth
[μ
m]
50
100
150
200
250X
Z
Polarization alteration due to linear optical propagation:
diattenuation, birefringence, polarisation scrambling
X
Y
α
Eω
Polarization-resolved SHG: calibration with rat-tail tendonsincident linear polarization at π/4 incident linear polarization
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Linear artefacts in SHG polarimetry
-π/2 -π/4 π/4 π/20α [rad] 0 100 200 300
100
80
60
40
20
0
SHG signal [arb. units]
z0
De
pth
[μ
m]
100
80
60
40
20
0
De
pth
[μ
m]
50
100
150
200
250
Fringes in intensity depth profile � birefringence:
0 100 200 300100
80
60
40
20
0
Polarization-dependent attenuation � diattenuation:
0 100 200 300100
80
60
40
20
0
Nonzero minima � polarization cross-talk (scrambling)
0 100 200 300100
80
60
40
20
0
100
80
60
40
20
0
-π/2 -π/4 π/4 π/20
Angle [rad]
De
pth
[μ
m]
0 0.5 1 1.5 2100
80
60
40
20
0
SHG signal [arb. units]D
ep
th [
μm
]
Experimental data
Gusachenko, Latour and Schanne-Klein, Opt. Express 18, 19349 (2010)Gusachenko, Latour and Schanne-Klein, Opt. Express 18, 19349 (2010)
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
x-talk, diatt., biref.uniform attenuation
Model calculations vs. experiments
Gusachenko, Latour and Schanne-Klein, Opt. Express 18, 19349 (2010)Gusachenko, Latour and Schanne-Klein, Opt. Express 18, 19349 (2010)
SHG / XSHG / X
0 0.5 1 1.5 2100
80
60
40
20
0
SHG signal [arb. units]
De
pth
[μ
m]
100
80
60
40
20
0
-π/2 -π/4 π/4 π/20
Angle [rad]
De
pth
[μ
m]
100
80
60
40
20
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Angle [rad]-π/2 -π/4 π/4 π/20
0 0.5 1 1.5100
80
60
40
20
0
SHG signal [arb. units]
100
80
60
40
20
0
-π/2 -π/4 π/4 π/20
Angle [rad]
De
pth
[μ
m]
50
100
150
200
250
0 100 200 300100
80
60
40
20
0
SHG signal [arb. units]
z0
De
pth
[μ
m]
le = 91 µm
lo = 190 µm
Experimental dataModel calculations
� Diattenuation: Δla = 134 µm
� Birefringence: Δn = 6.6 10-3
� Polar. Scrambling: 13 % at sample surface
� Ratiometric SHG response: ρ = 1.40
� Diattenuation: Δla = 134 µm
� Birefringence: Δn = 6.6 10-3
� Polar. Scrambling: 13 % at sample surface
� Ratiometric SHG response: ρ = 1.40
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
0 20 40 60 80 1000.7
0.8
0.9
1
1.1
1.2
1.3
1.4
1.5
ρ
Depth [μm]z0
Data processingExperimental data
Data processing for ρ and α determination
0 100 200 300100
80
60
40
20
0
SHG signal [arb. units]
z0
De
pth
[μ
m]
le = 91 µm
l0 = 190 µm
100
80
60
40
20
0
-π/2 -π/4 π/4 π/20Angle [rad]
De
pth
[μ
m]
0
40
80
120
160
0
30
60
90
120
150
180
210
240270
300
330
0
40
80
120
160
polarimetric diagram at
increasing depth
� α: fibrils orientation
� ρ: sub-µm organization
only diattenuation appears in the data processing
Gusachenko, Latour and Schanne-Klein, Opt. Express 18, 19349 (2010)Gusachenko, Latour and Schanne-Klein, Opt. Express 18, 19349 (2010)
Correction for
diattenuation
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Content
�Multiphoton microscopy� principle and setup
� Polarization-resolved SHG� principle
� linear artefacts in P-SHG
� Application to the tendon during
biomechanical assays
� Application to the cornea
� Conclusion and perspectives
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Fibril Lamellae
u
uuuβ
nanometer Collagen hierarchical structure
x
yz
xxxβ yyxβ
2≈=⇒yyx
xxxfib β
βρ
Y
X
Z
∑=cba
cbackbjaikji RRR ββϕθ
βχ ∑=kji
kjikKjJiIKJI RRR
triple helix
hyperpolarizability susceptibility
XXXχ XYYχ
YYX
XXX
χχ
ρ =
Cylindrical symmetryet et
micrometer
Collagen molecule
Non-linear optical physical quantities
Cylindrical symmetry
SHG tensorial response: susceptibility
ρ probes sub-µm heterogeneities
optical resolution
Within the tendon:
� angular dispersion of the fibrils at the
submicrometer scale
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
θ gaussian distribution,<θ>=0 - φ uniform
� polarization-resolved SHG probes sub-µm order
Tendon = model uniaxial tissue
Fascicle axis
Model calculation
0 10 20 30 40 50 60 701
1.5
2
2.5
3
σ [°], polar angle dispersion
ρ
ρfib=1
ρfib=1.36
ϕθ
βχ ∑=kji
kjikKjJiIKJI RRR
y
z
x
θφ X
Z
Y
O
)2(
)2(
yyx
xxx
χχ
ρ =
YYX
XXXfib β
βρ =
SHG tensorial response: susceptibility
I. Gusachenko et al, Biophys. J. (2012)
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Tendon biomechanics
Tissue deformations at
micrometric scale
controlled traction at
macroscopic scale
Monitoring polarization-resolved SHG response during mechanical assays
Goulam Houssen et al, J. Biomechanics, 44 (2011)
ρfib
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
SHG response at increasing strain
� SHG anisotropy ρ� Birefringence �
orientational order � orientational order �
� Fibrils disruption @ higher strains
� ρmin = ρfibril = 1.36
1st measure @ fibrils scale
Confirmation of our
theoretical approach
Good agreement with
previous data
Good agreement with
theoretical calculations
Cf Plotnikov et al, BJ 2006
Deniset-Besseau, JPCB 2009
Tuer et al, JPCB 2011
2 3 4 5 6 7 80
50
100
150
200
250
2 3 4 5 6 7 80
0.05
0.1
0.15
0.2
0.25
2 3 4 5 6 7 85
5.5
6
6.5
7
2 3 4 5 6 7 81.34
1.36
1.38
1.4
1.42
1.44
Strain [%] Strain [%] Strain [%] Strain [%]
ρ
Δn
[‰
]
η(0
)
Att
en
ua
tio
n le
ng
th [
µm
]
lo, le, Δlatt
Physiological
range
I. Gusachenko et al, Biophys. J. (2012)
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
� SHG anisotropy ρ� Birefringence �
orientational order � orientational order �
� Fibrils disruption @ higher strains
� ρmin = ρfibril = 1.36
1st measure @ fibril scale
Confirmation of our
theoretical approach
Good agreement with
previous data
Good agreement with
theoretical calculations
Plotnikov et al, BJ 2006
Deniset-Besseau, JPCB 2009
Tuer et al, JPCB 2011
2 3 4 5 6 7 80
50
100
150
200
250
2 3 4 5 6 7 80
0.05
0.1
0.15
0.2
0.25
2 3 4 5 6 7 85
5.5
6
6.5
7
2 3 4 5 6 7 81.34
1.36
1.38
1.4
1.42
1.44
Strain [%] Strain [%] Strain [%] Strain [%]
ρ
Δn
[‰
]
η(0
)
Att
en
ua
tio
n le
ng
th [
µm
]
lo, le, Δlatt
ρfib
SHG response at increasing strain
I. Gusachenko et al, Biophys. J. (2012)
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Content
�Multiphoton microscopy� principle and setup
� Polarization-resolved SHG� principle
� linear artefacts in P-SHG
� Application to the tendon during
biomechanical assays
� Application to the cornea
� Conclusion and perspectives
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Cornea SHG imaging: forward and backward
only backward signals are accessible for in vivo imaging…
How to retrieve the stromal structure
from the backward SHG signals ?
forward-SHG backward-SHG2PEF
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Polarization-resolved SHG: experimental setup
� Incident beam:
� circular or linear polarization
� orientation of the linear polarization
� Polarization-resolved SHG of both
F-SHG and B-SHG signals
2PEF
xy scanning
z
Ti-Sa
Laser
B-SHG
Filters
Dichroic
mirrors
y
x
λ/4
F-SHG
Filters
λ/2
Objective
Condenser
Power
adjustement
Cornea
10
20
30
40
50
0
30
60
90
120
150180
210
240
270
300
330
10
20
30
40
50
Acquisition
polarimetric diagrams
in each pixel of the
image
(angular steps : 10°)
Setup
� φ : orientation of the collagen fibrils
� ρ : anisotropy parameter
10
20
30
40
50
0
30
60
90
120
150
180
210
240
270
300
330
10
20
30
40
50
Data processing
CBA
CBA
+−++=ρ
CBAI yx +−+−=+ )(2cos)(4cos2 ϕαϕαω
x
Eωα
y
x
yXYφ
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Polarization-resolved SHG: application to cornea
Latour et al., Biomed. Opt. Express 3, 1-15 (2012)Latour et al., Biomed. Opt. Express 3, 1-15 (2012)
Modeling: CBAI yx +−+−=+ )(2cos)(4cos2 ϕαϕαω
� micrometric structures
� orientation of the
nanometric fibrils
forward SHG backward SHG
� orientation of the
lamellae even from the
epi-detected SHG signals
Orientation mapping of the collagen fibrils
Scale bar: 50 µm
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
In vivo polarization-resolved SHG imaging� Ex vivo eyeball imaging
porcine
eyeballScale bar: 50 µm
Latour et al., Biomed. Opt. Express 3, 1-15 (2012)Latour et al., Biomed. Opt. Express 3, 1-15 (2012)
� In vivo imaging
Scale bar: 50 µm
anesthetized
rat
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
φ and ρ variations through the stacked lamellae
20 22 24 26 28 30
0.6
0.8
1.0-90
-45
0
45
901.0
1.1
1.2
1.3
1.4
1.5
F-SHG B-SHG
Depth (µm)
F-SHG B-SHG
F-SHG B-SHG
ρtrans
φ
R²
� How to explain φ steep variations and plateaus / ρ minima?
� How reliable is our image processing in heterogeneous
microstructure (stacked lamellae)?
ρepi
ρ, φ and R² along few
lamellae (10 µm deep)ρ axial mapping within cornea
forward-SHG
backward-SHG
Scale bar: 20 µm
Scale bar: 20 µm
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Latour et al., Biomed. Opt. Express 3, 1 (2012)
Latour et al., Proc SPIE 8226-46 (2012)
Latour et al., Biomed. Opt. Express 3, 1 (2012)
Latour et al., Proc SPIE 8226-46 (2012)
20 22 24 26 28 30
0.6
0.8
1.0-90
-45
0
45
901.0
1.1
1.2
1.3
1.4
1.5
F-SHG B-SHG
Depth (µm)
F-SHG B-SHG
F-SHG B-SHG
ρ
φ
R²
Experimental results
φ and ρ variations : numerical calculations
60°
Numerical simulation
For each depth:
1.Simulation of the polarimatric
diagram
2.Modeling and determination
of ϕ and ρ
z
0 10 200
20
40
60 phi_fit rho_fit
Depth (arb. units)
phi (
°)
1.00
1.05
1.10
1.15
1.20
1.25
1.30
rhoρ
ϕ
Coherent summation of SHG
fields from 2 adjacent lamellae
0 10 20
-20
0
20
40
60
80
phi_fit rho_fit R²
Depth (arb. units)
phi (
°)
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
rho
ρ
R²
ϕ
Incoherent summation of SHG
radiations from 2 adjacent lamellae
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Latour et al., Biomed. Opt. Express 3, 1 (2012)
Latour et al., Proc SPIE 8226-46 (2012)
Latour et al., Biomed. Opt. Express 3, 1 (2012)
Latour et al., Proc SPIE 8226-46 (2012)
20 22 24 26 28 30
0.6
0.8
1.0-90
-45
0
45
901.0
1.1
1.2
1.3
1.4
1.5
F-SHG B-SHG
Depth (µm)
F-SHG B-SHG
F-SHG B-SHG
ρ
φ
R²
Experimental results
φ and ρ variations : numerical calculations
60°
Numerical simulation
z
0 10 200
20
40
60 phi_fit rho_fit
Depth (arb. units)
phi (
°)
1.00
1.05
1.10
1.15
1.20
1.25
1.30
rhoρ
ϕ
0 10 20
-20
0
20
40
60
80
phi_fit rho_fit R²
Depth (arb. units)
phi (
°)
0.6
0.7
0.8
0.9
1.0
1.1
1.2
1.3
rho
ρ
R²
ϕ
Coherent summation of SHG
fields from 2 adjacent lamellae
Incoherent summation of SHG
radiations from 2 adjacent lamellae
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Conclusion and perpectives (1)
Multiphoton imaging
� 3D
� without staining
� multimodal (2PEF and SHG)
Polarization-resolved SHG
� Experimental configuration in accordance with epi-detection
� Experiments and theory reveal polarization diagram distortion
� evidence of linear propagation effects :
birefringence, diattenuation and polarization scrambling
� χ(2)-tensor measurements must be corrected for diattenuation in
anisotropic media
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Conclusion and perpectives (2)
� Forward SHG:
striated structures � orientation of lamellae fibrils
� Backward SHG: polarimetry � orientation of lamellae fibrils
although raw images are spatially homogeneous
� In vivo polarization-resolved SHG
� characterization of 3D organization of the stroma
� optimization of polarization-resolved SHG setup and data processing
� characterization of corneal remodeling after injury or chirurgy
� mechanical assays on the cornea
macroscopic mechanical properties � microscopic structural organization
� tool of ophthalmic diagnosis ?
Application to tendon imaging
� Measurement of the anisotropy parameter ρ for a single fibril
� Coupling with mechanical assays: tissue deformation at the micrometer scale
� Follow-up of the sub-micrometric modification with polarization-resolved SHG
Application to cornea imaging (fibril diameter << resolution)
« Microscopie non-linéaire en sciences du vivant » – 15/10/2012Gaël LATOUR
Aknowledgments
Laboratory for Optics and Biosciences
I. Gusachenko
S. Bancelin
Y. Goulam Houssen
N. Olivier
A. Deniset-Besseau
I. Lamarre
E. Beaurepaire
M.-C. Schanne-Klein
latour@imnc.in2p3.fr
L. Kowalczuk
F. Aptel
K. Plamann
Laboratoire d’Optique Appliquée
Banque Française des Yeux (Paris)
I. Sourati
S. Gleize
P. Sabatier
A. Benoit
B. Lynch
J.-M. Allain
Laboratoire de Mécanique des Solides
References:Gusachenko et al., Opt. Express 18, 19349 (2010)
Gusachenko et al., Biophys. J. 102, 2220 (2012)
Latour et al., Biomed. Opt. Express 3, 1 (2012)
Latour et al., Proc SPIE 8226-46 (2012)
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