Osaka University	

Tomography of collagen fiber orientation inhuman tissue using depth-resolved secondharmonic-generation polarimetry

T. Yasui, Y. Hori, K. Sasaki, Y. Tohno*, and T. Araki
Grad. Sch. of Engg. Sci., Osaka Univ.
*Dept. of 1st Anat., Nara Medical Univ.
OWLS8 @ MELBOURNE

Osaka University	

Introduction
Collagen orientation

Mechanical and functional property of tissue and organ
Non-invasive, non-destructive
non-contact, non-staining△Conventional optical probe(scattering, absorption, fluorescence)

Collagen molecule induced

Second-Harmonic-Generation (SHG) light･Nonlinear interaction with ultrashort pulse light･Specifically generated from collagen molecule in tissue components･Sensitive to collagen orientation��SHG polarimetry!

!
!!

ref) !T. Yasui et al, J. Biomed. Opt. Vol. 9, pp.259-264 (2004).
!
T. Yasui et al, Appl. Opt., Vol. 43, pp. 2861-2867 (2004).

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Orientation vs. Polarization!
Axial(Non-centrosymmetric)

Collagenorientation

Cross-sectional(Centrosymmetric)

Parallel

Perpendicular

Collagen fiber

Collagen fiber

Collagen fiber

StrongWeakNoneLaser
polarization
SHG light

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Expectedorientation0

1.00.80.6
0.4
0.2
0.0900uniaxial

1.00.80.6
0.4
0.2
0.0900crossed
biaxial270

Human Achilles tendon

270

Collagen sponge

Human dentin
1.00.8
0.6
0.4
0.2
0.0

900

SHG intensity (a.u.)

900random270

SHG intensity (a.u.)

SHG intensity (a.u.)

1.0
0.8
0.6
0.4
0.2
0.0

SHG intensity (a.u.)90

SHG intensity (a.u.)

SHG intensity (a.u.)

1.0
0.8
0.6
0.4
0.2
0.0

SHG radar graph1.00.80.6
0.4
0.2
0.0900incompletelyrandom
incompletely
crossed270
270
uniaxial
biaxial270
Human anklebone Human reticular dermis
Human papillary dermis（across epidermis layer）	

Tissue dependence of collagen orientation

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Polarization-resolved SHG imaging @ human dentin
Polarization
angle

VerticalHorizontal

SHG Intensity(a.u.)

EnamelDentin

Localization of SHG lightOsaka University	

Polarization-resolved SHG imaging@ human Achilles tendon

100

intensity (a.u.)
SHGintensity
SHG(a.u.)

1.00
0.75

02mm

2mm

Polarization // Orientation

2mm

5050

0.50
0.252m257575m2mm2mm2mm

502mm

10010075SHG
anisotropy
PA value

SHGintensity
intensity(a.u.)(a.u.)
SHG

PA=0.86±0.062525000.002mm

2mm

2mm

Polarization ⊥ Orientation

Orientation degree

2mm

Uniform distribution of high PA values
Collagen orientation arranged with high organization

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Polarization-resolved SHG imaging@ collagen sponge
PA=0.07±0.05
100502502mm

Vertical polarization

75500.752mm

SHG intensity (a.u.)

751.00250.5000.25

PA value

SHG intensity (a.u.)

1000.002mm

Horizontal polarization

2mm

Orientation degreeUniform distribution of low PA values
Collagen orientation distributed equally in all directions

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@J@Polarization-resolved SHG imaging@ human talus

横配向	

骨陵	 (ｺﾗｰｹﾞﾝ、HOA)	 

1mm
光顕像	

縦配向	

骨陵に沿ったコラーゲン配向	Osaka University	

Microscopic 2D distribution of collagen orientation
2D mapping of orientation angle
angle @reticular dermis
Polarization-resolved SHG image
SHG intensity (a.u.)

測定領域：1mm*1mm
20µ mステップ

Area: 1mm*1mm

0500

100090deg
90deg

0deg
0deg
1.00.9

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.4

0.40.30.3

0.2

0.2

1mm

1.0

0.1

0.1

0.0

0.0
0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

0.0

1.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0120deg
120deg

30deg
30deg
1.0

1.0

0.9

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0.5

1mm

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

0.1

0.0
0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0.0
0.0

0.1

0.2

0.3

60deg
60deg

0.4

0.5

0.6

0.7

0.80.91.0150deg
150deg

1.0

1.0

0.9

0.9

0.8

0.8

0.7

0.7

0.6

0.6

0.5

0.5

0.4

0.4

0.3

0.3

0.2

0.2

0.1

Entangled structureof collagen fiber

0.1

0.0
0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0.0
0.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

Microphotograph of bovine dermis

Osaka University	

Introduction
Collagen orientation

Mechanical and functional property of tissue and organ
Non-invasive, non-destructive
non-contact, non-staining△Conventional optical probe(scattering, absorption, fluorescence)

Collagen molecule induced

Second-Harmonic-Generation (SHG) light����SHG polarimetry
Polarization-resolved SHG imaging

Present talk

Tomography of collagen fiber orientation in human tissues
based on depth-resolved SHG polarimetry

Osaka University	

Experimental setup
OL(x20, NA=0.42) HS
Sample(rotatory)
λ/2

pulse width=100fs, λc=800nm
rep.rate=80MHz, power=800mW

ML Ti:Sapphire laser
scanning
stagePLL

SHG, Laser(rotatory)

L(rotatory)
λ/2HSFsample

OCPPMTSHG lightNDPH(φ50µm)
Depth resolution= 14 µm

ANSig.

Ref.

Lock-in amplifier

Polarization condition
OCP; optical chopper, !ND; neutral density filter, P; polarizer, λ/2; half waveplate, HS;
harmonic separator, OL; objective lens, PH; pinhole, F; blue pass filter, A; analyzer,PMT; photomultiplierOsaka University	

Human tissue sample
Soft tissue

Hard tissue

Back dermis• formalin fixation, washing and drying• reticular layer• 1mm thickness

Dentin• sliced along tooth axis
• 1mm thickness

Achilles tendon• formalin fixation, washing and drying• sliced along axial direction• 2mm thickness

Anklebone• sliced across section• 1mm thickness

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SHG intensity (a.u.)

Penetration power of depth-resolved SHG measurement	
105104103102101100

Incident laser power = 20 mW

Human Achi lles
Human dermis
human de ntin

-1100Sample surface

100200300
Depth pos ition (µm)

400500Osaka University	

Basic performance of depth-resolved SHG polarimetry

12060300(H-PL)

210

330
240

27030010.80.60.40.20120(V-PL)
9060300(H-PL)

210

SHG intensity(normalized)

10.80.60.40.20(V-PL)
90

SHG intensity(normalized)

SHG intensity(normalized)

Expected collagen orientation
10.80.60.40.20330
240

270300120(V-PL)
9060300(H-PL)

210

330
240

270300Depth=0µm(suface of 1st layer)

Depth=300µm(boundary between 1st and 2nd layers)

Depth=350µm(2nd layer)

Horizontal
Uniaxial

Mixture of horizontal and vertical

VerticalOsaka University	

Basic performance of depth-resolved SHG polarimetry

12060300(H-PL)

210

330
240

27030010.80.60.40.20120(V-PL)
9060300(H-PL)

210

SHG intensity(normalized)

10.8
0.6
0.40.20(V-PL)
90SHG intensity(normalized)

SHG intensity(normalized)

Expected collagen orientation
10.8
0.6
0.4
0.20330240270300120(V-PL)
9060300(H-PL)

210

330
240

270300Depth=0µm(suface of 1st layer)

Depth=300µm(boundary between 1st and 2nd layers)

Depth=350µm(2nd layer)

Horizontal
Uniaxial

Mixture of horizontal and vertical

VerticalOsaka University	

Depth-resolved SHG radar graph of human tissue	

360

300300

Polarization angle (deg)

360

240
180120600Sample
surface80160240

3204001.00.5240018012060000102030405060708090

Depth position (µm)

Depth position (µm)

Human Achilles tendon

SHG intensity (normalized)

Polarization angle (deg)SHG radargraph

Samplesurface

Human dentin

Uniform distribution of collagen orientation along depth direction

Osaka University	

Depth-resolved imaging of 2D lateral distribution ofcollagen orientation in human anklebone
Orientaion degree
IV - IH
ORD = I + IVH2.5mm

IV; vertically-polarized SHG intensity
IH; horizontally-polarized SHG intensity
Uniaxial for ORD=±1Random or Biaxial for ORD=0-0.52.5mm

Depth = 50µm

0Horizontal

0.5

VerticalDepth = 0µm(sample surface)

Depth = 100µm

Depth = 150µm

Collagen orientation parallel to bone crest direction
developing along depth direction

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In vivo measurement of human skin
Scan direction

SHG intensity (a.u.)

Volunteer’s forehand

1.5*1051.0*1055.0*10400tapeskin

2468Transverse position (mm)tape1012Osaka University	Summary(1) Depth-resolved SHG polariemtry•Depth resolution = 14 µm•Penetration power = 100µm@human hard tissue150~300µm@human soft tissue(2) Tomography of collagen orientation in human tissue•Depth-resolved SHG radar graph•Depth-resolved imaging of 2D lateral distribution ofcollagen orientation(3) In vivo measurement of human skin