ftiri provides a “chemical photograph” in thin sections of tissue the tissue must be...
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FTIR Imaging - Introduction
•FTIRI provides a “chemical photograph” in thin sections of tissue
•The tissue must be sufficiently thin to allow the passage of infrared radiation (1-2 um for bone, 2-5 for cartilage, skin and tendon.
•The process is non-destructive in that there are no dyes or other treatments, thus the thin sections can be reused for other applications.
•This tutorial will describe parameters validated for bone and dentin and provides examples of the types of questions that might be asked.
•For other applications please contact the core technician or director.
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PMMA
AMIDE IAMIDE II
PO 43-
1700 1600 1500 1400 1300 1200 1100 1000 1700 1600 1500 1400 1300 1200 1100 1000
Wavenumber (cm-1)
1a
1b
0
1.000
2.000
3.000
4.000
5.000
1c
0
0.2000
0.4000
0.6000
0.8000
1.000
1d
4.75.87.08.19.210.4 11.6
Wt % CO2-
0.150.170.190.210.230.250.270.290.310.330.350.370.390.41
3Mineral Mineral:Matrix
CrystallinityCarbonate
Chemical Photography of Bone Composition
CO3=
PMMA
AMIDE IAMIDE II
PO 43-
1700 1600 1500 1400 1300 1200 1100 1000 1700 1600 1500 1400 1300 1200 1100 1000
Wavenumber (cm-1)
CO3=
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PMMA
AMIDE IAMIDE II
PO 43-
1700 1600 1500 1400 1300 1200 1100 1000 1700 1600 1500 1400 1300 1200 1100 1000
Wavenumber (cm-1)
1a
1b
0
1.000
2.000
3.000
4.000
5.000
1c
0
0.2000
0.4000
0.6000
0.8000
1.000
1d
4.75.87.08.19.210.4 11.6
Wt % CO2-
0.150.170.190.210.230.250.270.290.310.330.350.370.390.41
3Mineral Mineral:Matrix
CrystallinityCarbonate
Chemical Photography of Bone Composition
CO3=
PMMA
AMIDE IAMIDE II
PO 43-
1700 1600 1500 1400 1300 1200 1100 1000 1700 1600 1500 1400 1300 1200 1100 1000
Wavenumber (cm-1)
CO3=
Lipids Sugars
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First Report 1998Cell Mol Biol (Noisy-le-grand). 1998 Feb;44(1):109-15.Infrared microspectroscopic imaging of biomineralized tissues using a mercury-cadmium-telluride focal-plane array detector.Marcott C, Reeder RC, Paschalis EP, Tatakis DN, Boskey AL, Mendelsohn R.AbstractA 64 x 64 mercury-cadmium-telluride focal-plane array detector attached to a Fourier transform infrared microscope was used to spectroscopically image 5 microm sections of canine alveolar bone tissue in the fingerprint region of the infrared spectrum. By ratioing the relative intensities of specific bands across the images, it is possible to obtain spatial distributions of the mineral-to-matrix ratio and mineral maturity as a function of distance from an osteon.
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Validated FTIRI Parameters
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Mineral:Matrix Ratio
FTIR mineral:matrix ratio correlates with ash fraction
0 10 20 30 40 50 60
Spectroscopic Mineral:Matrix Ratio
0.00
0.20
0.40
0.60
0.80
1.00
Fra
cti
on
al
As
h W
eig
ht
r2 = 0.83p < 0.05
Mineral:Matrix Ratio is Linearly Related to
Ash Weight (Mineral Content)
FTIR Mineral:Matrix Ratio
Ash
Fra
cti
on
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Mineral Crystallinity
FTIR crystallinity correlates with c-axis length assessed by x-ray diffraction
1030:1020 Peak Area Ratio Reflects Crystal Size
0.50 1.00 1.50 2.00 2.50 3.00
1030/1020 Area Ratio
130
140
150
160
170
180
c-A
xis
Th
ickn
ess (
A)
R2=0.98
FTIR Crystallinity
c-a
xis
dim
en
sio
n
(Ǻ)
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Carbonate:Phosphate Ratio Validated
Chemically
Magne et al., Bone, 2001
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Collagen Maturity (XLR)
Paschalis et al., Bone, 2011
Whole bone Chemistry Bone surface FTIRI
Cortical Periosteal surface
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FTIRI Parameters Correlate with
Mechanical Properties
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Vertebral bone strength correlates with vertebral
crystallinity
0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.215
20
25
30
Str
ess (
MP
a)
1030/1020 (area ratio)
R2= 0.29 p= 0.03
Weinstein et al., Aging Cell, 2010
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Synchrotron Data
Judex et al, Calcif Tissue Int, 2005
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Bone Volume 47, Issue 6 2010 1030 - 1038
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What is needed to perform FTIRI of Bone
FTIRI is a transmission experiment = thin sections
Imaging Spectrometer Software for data processing
Embedding media: PMMA, Spurs, LR-white
Zebra fish and calvaria, cell cultureScales
D16, 4P
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Boskey et al, BBA, 2006
EFFECT of EMBEDDING MEDIA
PMMA, Spurr, but not GMA can be removed by spectral subtraction.
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(Donnelly et al., JOR, 2011)
•Mean values of all parameters similar across sites (a,b). •Heterogeneities more variable across sites (1c,d). •In trabecular tissue, the heterogeneity of the crystallinity at the sub-trochanter was greater than that of the iliac crest (+46%, p=0.008) and the greater trochanter (+36%, p=0.019).•No differences in heterogeneities of other trabecular tissue properties, nor in heterogeneities of cortical parameters.
Values normalized to those at the iliac crest. Missing bars indicate that the tissue type was not present in the biopsy, n=4
Tissue Heterogeneity:Select a Single Tissue for Comparison
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Processing FTIRI Data
Subtract contribution of embedding media and water vapor
Display raw images Univariate analysis
Mapping parameters Curve fitting and deconvolution
Multivariate analysis PLS, Cluster Analysis, Factor Analysis
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Table I
Table I: Infrared Parameters used in Imaging of Bone Parameter Wavelength Range (cm-1) Validation Correlates with Mineral:Matrix 900-1200/1585-1720 Ash Wt. 6 BMD and ash weight, fracture
risk18,19 Carbonate:Phosphate 850-890/900-1200 Chemical
analysis15 Age (increase)20
Collagen Maturity 1660/1690 Chemical analysis16
Pyridinoline/deoxypyridinoline; fracture risk19
Crystallinity 1030/1020 X-RD6 Bone strength, fracture risk21 HPO4 substitution 1128/1096 Model
compounds17 Bone age (inverse)17
References available on request
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Applications : Answering Specific Questions about Bone
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Effect of Tissue Age: Healthy Baboon Study
4
4.5
5
5.5
6
6.5
7
Pixels
Pix
els
10 20 30
5
10
15
20
25
30
35
40
Mineral to Matrix Ratio
1.2
1.25
1.3
1.35
Pixels
Pix
els
5 10 15 20 25 30 35
5
10
15
20
1.2
1.25
1.3
1.35
Pixels
Pix
els
5 10 15 20 25 30 35
5
10
15
20
1.2
1.25
1.3
1.35
Pixels
Pix
els
5 10 15 20 25 30 35
5
10
15
20
1.2
1.25
1.3
1.35
Pixels
Pix
els
5 10 15 20 25 30 35
5
10
15
20
1.2
1.25
1.3
1.35
Pixels
Pix
els
5 10 15 20 25 30 35
5
10
15
20
10%50%
100%
SHG
Gourioon-Arsiquaud et al JBMR, 2010
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Mineral/Matrix, Crystallinity & CO3/Amide I
2.5
R2 = 0.8566
R2 = 0.8328
R2 = 0.8969
3.5
4
4.5
5
5.5
6
0 5 10 15 20 25 30
Baboon Age (years)
Min
/Mat
rix ra
tio
10% 20% 30% 40% 50% 60% 70% 80% 90% 100%
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Collagen Cross link Ratio (XLR)
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FTIR
Interstitial Tissue
Secondary Osteon
Old Osteon
Age Dependent Changes in Mature Human Bones
Cadaveric Femurs
Male (n = 12)
Middle-aged (n = 6)
Old(n = 6)
Secondary Osteons x 3Old Osteons x 3
Interstitial x 3
M. Reyes, Dissertation, U Tx San Antonio
Second ost eoO l d O st eonI nt er st i t i al Second ost eoO l d O st eonI nt er st i t i al0
1
2
3
4
5
6
7
Mi n e ra l /Ma tri x
Mi d d l e Ag e d Ol d
Cadaveric Femurs
Interstitial tissue has higher Min/Mat than secondary or old
osteons
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Can FTIR Parameters Predict Fracture Risk Independently of BMD? Collagen Maturity Crystallinity
Crystallinity[Gourion-Arsiquaud et al. 2009]
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FTIRI Parameters Predictive of Fracture Risk – Iliac Crest BiopsiesGlobal averages – all parameters
* p<0.05; **p<0.001; n=52
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Multiple logistic regression model
Fracture (0/1) = bo +b1BMD +b2age +b3 Rx +b4(min/mat) +b5(xstl) + b6(XLR) + b7(CO3/P)
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Studies of KO animals: DMP-1 KO
Min//Mat
XST
Ling et al., JBMR 2005
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Studies in mutant animals: models where collagen synthesis is defective
fro/fro WT
Mineral/Matrix
0
4
8
12
fro/fro oim/oim
-/-
+/+
Crystallinity
0
0.5
1
1.5
fro/fro oim/oim
-/-
+/+
Carb/Phosphate
0
0.004
0.008
fro/fro oim/oim
-/-
+/+*
*
Coleman, et al. Bone, 2012
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0
1
2
3
4
5
6
7
Placebo ALN
Effects of Treatment P
ara
mete
r M
ean
Boskey et al., 2009
M/M C/P C/Am I Xst XLR M/M C/P C/Am I Xst XLR0
1
2
3
4
5
6
7
*
**
Cortical Bone Cancellous Bone
*
Mineral/Matrix significantly increased in women without fractures treated 2 yrs with ALN
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Compositional Heterogeneity Healthy bone has a broad distribution of
tissue properties reflecting the presence of new and old bone
A heterogeneous tissue may be better able to resist crack propagation [Burr & Martin 1986]
4.555.566.50
50
100
150
200
250
300
350
Nu
mb
ers
of
Pix
els
FWHM
A)
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M/M C/P C/Am I XLR XST M/M C/P C/Am I XLR XST
Cortical Cancellous
0
1
2
3
4
5
6
FWH
MPlacebo
*
*
* *
*
*
**
ALN
Heterogeneity is Decreased in the ALN treated patients
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Multivariate Analysis: PLS, Factor Analysis, Cluster Analysis, Neural Networks
Factor Analysis
F1
F2
0.982
0.984
0.986
0.988
0.99
0.992
0.994
0.996
0.998
20 40 60 80 100 120 140 160
20
40
60
80
100
120
140
micrometers
mic
rom
eter
s
F3 F6
F5
F4
10001100
F1
F6
Factor Loadings
Spevak et al., Calcif Tissue 2013
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Future DirectionsSpecular Reflection (SR)
Reflection from a mirror
Nicholson et al., Anal Chem 2012 84(7) 3369.
Racehorse calcified cartilage and subchondral bone (uniform age) at potential sites of fracture
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Future Directions – AFM IR to be acquired July 2014
© 2012 Anasys Instruments
800100012001400160018000
5
10
15
20
25
30
35
15μm40μm
nanoIR Spectra Collected at Different Distances from Osteon Center
AFM image 60 x 5 μm