University of Tokushima	Collagen-sensitive
microscopy
Takeshi Yasui
Inst. Tech. Sci., Univ. Tokushima, Japan
Grad. Sch. Engg. Sci., Osaka Univ., Japan
August 2, 2012

University of Tokushima	To search “collagen” on the internet	
Collagendrinks	Collageninjections	Collagen
supplements	

CollagensoupCollagen cream	• Important in body
• Huge market in world(¥55 B in Japan@ 2011)• Questionable ?	

University of Tokushima	

What is collagen?	 

Cell	

Collagen plays arole of “glue” tobind cells together
or makes partitions
between cells toput them in theright and wellCollagen plays anCollagenordered position	  active role in biding and(extracellular matrix)	
supporting cells!	
• Collagen makes up every part of body and supports, binds, detaches cells• Collagen is a scaffold for cells. Cells divide and proliferate there

Collagen makes significant influence onvital activity and aging	University of Tokushima	

What is collagen?	 

collagen	

water	

Others	Collagencontent indry weightof skin	Collagencontent indry weightof tendon	Collagencontent indry weightof bone	• Skin loses firmness and elasticity, and sags andWhathappens as wrinkles appears
collagen • Bone density decreases causing osteoporosis• The gums deteriorate and periodontal disease occurs
production • Blood vessels lose elasticity causing hypertension
decreases?	
• Nails become liable to split or chipRef) http://www.hopec.biz-web.jp/collagen/collagentoha/University of Tokushima	

What is collagen?	

•Most abundant fibrous protein; principal
component of ECM•Produced and remodeled by fibroblast•Scaffold to maintain tissue structure	
•Control production, specialization, andmorphogenesis of cells	
nPlay an important role as a scaffold forcells in engineering tissues	Hierarchical structure of collagen 	

Fiber	

1 µm	0.5 - 3 µm	

CollagenFibril	Tropocollagen	

Fibroblast	

Polypeptide chain	

10- 300 nm	

1.5 nm	Triple-helixstructure	University of Tokushima	Collagen in skin	 

Epidermis

DermisSubcutaneous
http://www.i-nfc.com/newpage2.htm
Collagencontent = 70wt%• Collagen comprises 70%
of dermis• Collagen determines
mechanical andfunctional characteristics
of skin• Collagen draws attention
in the field of skincosmetics and anti-aging
dermatology
http://www.i-nfc.com/newpage2.htmより抜粋

University of Tokushima	

Conventional method to observe collagenStaining method

Electron microscopy

Selective visualization
Invasive (skin biopsy)	

Highly spatial resolution
Invasive (skin biopsy)	

Difficult to visualize distribution ofcollagen in living tissue “in vivo”

University of Tokushima	

Optical probe methods

Laser confocal microscopy	Structure of collagen fiber and bundle in dermis
Collagen content in dermis : 70 wt%• Simpleand rapid
Mechanical and functional property of skin
• Non-contact
and non-invasive! (tension, elasticity, and water content)• Directlyto theburn,skinand etc)
Skinapplied
disease (cancer,Skin cosmeticin vivo
measurement
In vivo optical probe for collagenstructure(non-invasive, non-contact, non-staing,and 3D imaging)No selectivity
to collagen	

Collagen molecule induced

Second-Harmonic-Generation (SHG) lightNonlinear optical interaction with ultrashort pulse lightSpecifically generated from collagen molecule in tissue components
Sensitive to collagen structure and orientation!
!

ref) T. Yasui et al, J. Biomed. Opt., Vol. 9, pp. 256-264 (2004).
T. Yasui et al, Appl. Opt., Vol. 43, pp. 2861-2867 (2004).

University of Tokushima	

What is SHG light?!
LinearNonlinearSound(freq. = f)

f

Touch

Strong Hit!

2f

Sound(Keyboard)

CW light

ωLight

Reflected andscattered lightω

Non centrosysmetric materialω

SHG light2ω
Ultraintense
pulse lightω
w
Non centrosysmetric materialex) anisotropic crystal, tissue collagenUniversity of Tokushima	

What is femtosecond (10-15 sec) ?
sec秒

Time
時間

1s

ミリ
ms

10-3µsマイクロps
ピコ

ナノns

10-6

10-9

フェfs
ムト
10-15

10-12

♥
スパコン
１命令
Super
computer	

心臓の鼓動
Heart
beating	

トランジスタTransistor	
動作限界

分子の運 動Molecular vibration	

散乱Electronic電子のscattering	
光の周期

Oscillation of light	

光の進む3,000km
30万km

300km

300m

30cm

0.3mm

0.3µm

Distance
距離

地球を7 .5周
Go aroundearth
7.5-times	

人の身長Man	

ウィ ルス

Virus	

University of Tokushima	

CW (continuous-wave) light vs. fs pulse light!
CW (continuous-wave)
laser!
連続光
時間、空間
Time,
space

fs超短パルス光
pulse laser!

Average power is equal to each other.

12ns (87MHz)

Electric filed of light is
temporally localized withinregion of fs order.

(3.6m)
60fs(18 µm)〜
Optical nonlinear effectcan be easily induced
without thermal damage!

Ultrahighpeak power
時間、空間
Time,
space

University of Tokushima	Various femtosecond lasers in our lab	“Lab-made” femtosecond Ti:Sapphire laser �at wavelength of 800 nm�
“Amplified” femtosecond Ti:Sapphire�laser at wavelength of 800 nm�

¥ 8M	
“Compact” femtosecond fiber laser �at wavelength of 1550 nm�

¥ 5M	

¥ 29M	
“Penetrative” femtosecond�
Cr:Forsterite laser �at wavelength of 1250 nm	

¥ 10M	
Good forbiomed. opt.	

University of Tokushima	

Structure of tissue collagen!
tropocollagen(triclinic structure)

microfibril
fibrilfiber

Non centrosymmetric structure

Generation ofbundleSHG light
triple-twisted
polypeptide helix
Uniformly-ordered, higheruniformly-ordered
orderedstructures
orienattion
measured area
Coherently enhanced
φ=10µmEnhanced
SHG lightand polarized
SHG duelighttoconstructive interferenceUniversity of Tokushima	Collagen-sensitive SHG microscopy
for dermatological applications

Selective visualizationof collagen fiber in vivo

University of Tokushima	

Experimental setup	λc = 1250nm
Δt = 100 fs
frep = 73 MHz
Pavg = 250 mW	

Confocal
image

SHG
image	

Image acquisition time:
2 sec@600µm*600µm

University of Tokushima	

SHG imaging of sliced specimen	 
Image size
400µm*400µm	 

Uniformorientation
Denseof thick
distribution
collagenof thinfiber	  point is thatcollagenImportant
Tail tendon 	 
Papillary dermis	 fiber	 only collagen fiber in
Partially
Entangles
tissueisvisualized!	
entangles
structure
structureof thickof thickcollagencollagenfiber	 fiber	 Upper reticular dermis	 
Lower reticular dermis	 University of Tokushima	

Large-area SHG imaging(Image area: 2.4 mm×2.4 mm)
Image area
(600µm*600µm)	
Obtained by arranging 16
SHG images(600µm*600µm) as a matrix
of four rows and four lines.
Image acquisition time:1minUniversity of Tokushima	

Optically-sectioning, depth-resolved SHG imaging	 
Sample: frozen porcine skin
Area：400 µm×400 µm
Pixel number:256×256
Acquisition time: 10 sec/ image
laser power = 10mW

ProbingplaneEpidermis
Dermis	Dense distribution of fine collagen fiber

Capillaries

Thickly growing collagen fiberUniversity of Tokushima	

in vivo depth-resolved SHG imaging of human forearm
58-years malearea: 600×600mm
time: 2 sec/image

Measured area

0 µm	10 µm	

20 µm	

30 µm	

40 µm	

50 µm	

60 µm	

70 µm	

80 µm	

90 µm	

100 µm	110 µm	120 µm	

130 µm	

140 µm	150 µm	

160 µm	

170 µm	

180 µm	

190 µm	

200 µm	

210 µm	

220 µm	

230 µm	

240 µm	

250 µm	

260 µm	

270 µm	

280 µm	

290 µm	

300 µm	in vivo large-area SHG imaging ofhypertrophic scar in human forearm

University of Tokushima	(image size =4.4mm*4.4mm)

Abnormal structure of collagen fiber caused byimperfect healing process of injury

Summary

University of Tokushima	

Tissuesstructure	Collagendensity	Collagenorientation	

in vivo visualization of “living” collagen	Burn assessment

Anti-aging
Photoaging, intrinsic aging
Wrinkle formation	

Burn depth, scarformation, skin grafting	

Wound healing	
Dynamics of collagen
during healing process	

Tissue engineering
Quality control of collagenin engineered tissues