SECOND-HARMONIC GENERATION IMAGING OF
COLLAGEN-BASED SYSTEMSpresented by
Dr. K. C. Toussaint, Jr.Laboratory for Photonics Research of Bio/nano
Environments (PROBE)Dept. of Mechanical Science and Engineering
2010 Nano-Biophotonics Summer SchoolUniversity of Illinois at Urbana-Champaign
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LECTURE OUTLINE
Second-harmonic generation review
Application of SHG imaging to qualitative studies
Application of SHG imaging to quantitative studies
Fourier Transform-SHG microscopy
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REVIEW: NONLINEAR MICROSCOPY
• Nonlinear methods – approaches whereby output intensity is proportional to In, where I is the input intensity and n is the number of photons involved in the interaction
• Permits “optical histology”
• Deeper penetration depths(~600 µm compared to 50 µm)
• Reduced photodamage
• Reduced photobleaching Denk et al, Nature Methods 2, 932 - 940 (2005)
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REVIEW: BASIC CONCEPT
• SHG, THG “no energy” is absorbed• 2PF energy is absorbed
Image Source: Handbook of Biological Confocal Microscopy
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REVIEW: SHG IN BIOLOGY
• Applies to noncentrosymmetric systems, i.e., those that are highly ordered (spatially organized)
• Examples in biology include proteins: collagen, myosin, and tubulin
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COLLAGEN• Accounts for 25% of total protein mass in mammals
• Molecule is 1.5-nm width, 300-nm length
• Fibrillar collagen found in connective tissues
• Displays high degree of (supramolecular) organization
• Provides tensile strength in bones, transparency in cornea, elasticity in skin
http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mcb.section.6542
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COLLAGEN (TENDON EXAMPLE)
Length of
300 nm
10 µm
10 mm
Fratzl, P., Current Opinion in Colloid and Interface Science 8, 32-39 (2003)
- Gap (G) contains 4 collagen molecules
- 3D arrangement is not well understood
Tendon consists of mostly type I
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COLLAGEN AND SHG
• SHG has definitely been observed for Type I, II, III (w/ I the most crystalline)
• Form 90% of types found in body
• Used to study (normal and abnormal) structure in tendon, skin, cornea, bone, etc.
• Occasionally SHG imaging combined with other modalities to increase sensitivity
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QUALITATIVE APPLICATION OF SHG MICROSCOPY
• Studied collagen fiber organization in corneal stroma between human, mice, and rabbits
• Observation of bands of fibers (lamellae) in forward SHG
• Observed 2 types of lamellar organization common in all species in anterior (interwoven) and posterior (orthogonal)
• Only human showed transverse lamellae that may serve anchoring role
Human Mouse Rabbit
A
P
50 µm
N. Morishige et al, J. Cataract Refract Surgery 32, 1784 (2006)
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QUALITATIVE APPLICATION OF SHG MICROSCOPY
• SHG combined with OCT and histopathology to study wound healing in skin-equivalent tissue models (composed of type I collagen)
• Tissue exposed to thermal (laser-induced) injury
• OCT used to monitor reduction in (linear) light scattering; SHG used to monitor reduction in collagen organization
A. Yeh et al, J. Biomedical Optics 9, 248 (2004)
OCT
SHG
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QUALITATIVE APPLICATION OF SHG MICROSCOPY
• SHG combined with 2PF to distinguish between various scaffold materials used in tissue engineering
• Monitored difference in emission spectrum and SHG intensity
• Alternative to using histology (and staining) for examination
open-cellpolylactic acid
polyglycolicacid
Y. Sun et al, Microscopy Research and Technique 71, 140 (2008)
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QUALITATIVE APPLICATION OF SHG MICROSCOPY: CLINICAL USE
JenLab GmbH
Konig et al, Journal of Biophotonics 1, 506 (2008)
- Potential for clinical use
- Development of high-NA GRIN opticspermits high-resolution imaging
-Combined with 2PF microscopy
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WHAT ABOUT QUANTITATIVE ANALYSIS?
• Why do we care?
• Attempt to model/quantify changes in collagen fibril organization due to physical injury or disease (lupus, Marfan syndrome, assess collagen content in tumors)
• Need to develop metrics to use as markers (correlate with morphology/physiological function)
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APPLICATION OF SHG MICROSCOPY: QUANTITATIVE
• Used the ratio of forward-to-backward SHG (F/B) to study tendon collagen
• Deduced that SHG emanates from fibril shell rather than from its bulk
• F/B ratio sensitive to ionic strength of solution; results in change in shell thickness
R. Williams et al, Biophysical Journal 88, 1377 (2005)
F B
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APPLICATION OF SHG MICROSCOPY: QUANTITATIVE
• Morphological changes are not observable from images
• Obvious changes in contrast is indicative of sensitivity to ionic concentration
• Fibril shell thickness is believed to “thin” with increasing ionic concentration
R. Williams et al, Biophysical Journal 88, 1377 (2005)
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APPLICATION OF SHG MICROSCOPY: QUANTITATIVE
• Developed metrics for disorder in colllagenous tissue using polarization-modulated SHG
• Disorder index
• Looked at mouse model of intervertebral disk injury
• Orientation of fibers is estimated by modulating input polarization
Maximally ordered
Disordered
K. Reiser et al, Journal of Biomedical Optics 12, 064019 (2007)
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APPLICATION OF SHG MICROSCOPY: QUANTITATIVE
Control disk
Disk after compressive loading
K. Reiser et al, Journal of Biomedical Optics 12, 064019 (2007)
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POLARIZATION• Often polarization microscopy is used on the same
samples as SHG microscopy
• Contrasts are not the same
• Polarization microscopy is based on linear birefringence
• SHG microscopy depends on nonlinear dependence on input power
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POLARIZATION IN SHG IMAGING
• Used to determine matrix elements
• Provides info on degree of organization of the molecular dipoles via anisotropy parameter
• Extract orientation information
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SHG AND ANISOTROPIC MATERIALS
.....3)3(2)2()1( +++= EEEP χχχ
Induced polarization
.
)()(2)()(2)()(2
)()()(
)2()2()2( 2
2
2
363534333231
262524232221
161514131211
3
2
1
=
ωωωωωω
ωωω
χχχχχχχχχχχχχχχχχχ
ωωω
xy
zx
yz
z
y
x
EEEEEE
EEE
PPP
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NONCENTROSYMMETRICCRYSTAL CLASSES
Image Source: Nonlinear Optics (2nd Edition)
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APPLICATION OF SHG MICROSCOPY: QUANTITATIVE
• Polarization degree of freedom was used
• Ratio of χ(2) elements used as a metric
• Different tissues had different collagen types (I and III)
F. Tiaho et al, Optics Express 15, 12286 (2007)
Frog tendon
10 µm
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0000000000000
333131
15
15
dddd
d
F. Tiaho et al, Optics Express 15, 12286 (2007)
E
y
xz
α
Col
lage
n
Determined “effective” orientation of collagen harmonophores
APPLICATION OF SHG MICROSCOPY: QUANTITATIVE
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APPLICATION OF SHG MICROSCOPY: QUANTITATIVE
• Again, ratio of χ(2) elements used as a metric
• This time, χ(2) determined as a function of space
• Looked at human dermis and tendon-muscle junction of chicken wing
W. Chen et al, Applied Physics Letters 94, 183902 (2009)
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APPLICATION OF SHG MICROSCOPY: QUANTITATIVE
• Histogram distribution for χ(2)
for human dermis suggests detection of type III collagen in addition to type I
W. Chen et al, Applied Physics Letters 94, 183902 (2009)
Type IType III
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APPLICATION OF SHG MICROSCOPY: QUANTITATIVE (FOURIER ANALYSIS)
• SHG is excellent for imaging fibrous structures
• Fourier transforms is a useful tool for analyzing fibers/fiber arrays
• Some information about spatial organization can be obtained without the use of complex algorithms or experimental setup
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Fhigh
Dominant frequencies
Magnitude Spectrum
• Fhigh and peaks in M.S.
info about min. observable
feature size and collagen
fiber packing (spacing)
• Evaluated for direction of
preferred orientation
FT-SHG: SPATIAL FREQUENCY
Spatial Frequency
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Orientation = 80° Orientation = 170°-90°=80°
FT-SHG: ORIENTATION
• FT picks up preferred direction of variation in image
• Creation of binary image of dominant spatial frequencies apply best-fit line
FT
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5 10 15 20 25 30 35
5
10
15
20
25
30
35
54.8°±0.6 °
170.38°±0.3°
55° 90°
171°
5 10 15 20 25 30 35
5
10
15
20
25
30
35
90°±0 °
5 10 15 20 25 30 35
5
10
15
20
25
30
35
(a) (b)
FT-SHG: ORIENTATION
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GALVOSCANNER
SCAN LENS
TUBE LENS
TI:SAPPHIRE
SAMPLE
LASER BLOCKING FILTER
SHG FILTER
OBJECTIVE 1
DETECTOR
OBJECTIVE 2
EXPERIMENTAL SETUP
R. Ambekar et al, Optics Express 17, 14534 (2009).
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69.4°±4.0°
(a) (b)
71.3°±3.0°
69.0°±1.6°
1
2
3
1 2
3
1
2
3
FT-SHG: ORIENTATION
R. Ambekar et al, Optics Express 17, 14534 (2009).
Porcine Ear Cartilage
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107.8°±7.6°
(a) (b)
45.5°±5.2°
51.8°±3.5°
4
5
6
4 5
6
FT-SHG: ORIENTATION
R. Ambekar et al, Optics Express 17, 14534 (2009).
Porcine Trachea Cartilage
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0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.60
5
10
15
20
CO
UN
TS
Fhigh [µm-1]
EAR
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.60
5
10
15
20
CO
UN
TS
Fhigh [µm-1]
TRACHEA
(a)
(c)
(b)
(d)EAR TRACHEA
~1.3 to 2 µm ~.7 to 3 µm.
FT-SHG: SPATIAL FREQUENCY
~3 µm
R. Ambekar et al, Optics Express 17, 14534 (2009).
More consistency in structure
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TENDON SCLERA EAR CARTILAGE
DE
PTH
0
µmD
EPT
H 1
0 µm
DE
PTH
20
µm
A
BC
BWA
BC
FW
FW
BW
FW
BW
FW
BW
FW
BWFW
A
B
C
A
B
C
A
B
C
A
BC
A
BC
A
BC
A
BC
BWFW
BWFW
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
A
B
C
BWFW
BWFW
A
B
C
A
B
C
A
B
C
FT-SHG: FORWARD VS. BACKAWARD
R. Ambekar et al, Optics Letters 34, 3779 (2009).
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FT-SHG: FORWARD VS. BACKAWARD
R. Ambekar et al, Optics Letters 34, 3779 (2009).
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SUMMARY• SHG is a nonlinear optical technique (coherent
scattering)
• Useful in microscopy for non-invasive 3D imaging
• Recent work has focused on combining with other nonlinear imaging modes
• Development of quantitative metrics could help with assessment of tissue morphology
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ACKNOWLEDGEMENTS
• NSF DBI-0839113• NSF CAREER Award
Raghu Ambekar Rao Monal Mehta
http://probe.mechse.illinois.edu
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