Microscopie non-linéaire résolue en polarisation pour l...

« 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


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


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)


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)


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


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


Content









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


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


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


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


Linear artefacts in SHG polarimetry

-π/2 -π/4 π/4 π/20α [rad] 0 100 200 300

100

80

60

40

20

0


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)


x-talk, diatt., biref.uniform attenuation

Model calculations vs. experiments


SHG / XSHG / X

0 0.5 1 1.5 2100

80

60

40

20

0


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


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


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


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


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


Correction for

diattenuation


Content









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


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)


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


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



� 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



Content









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


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


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


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


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


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






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


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


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)


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

[email protected]

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)



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