Transcript Infrared Imaging of Bone - Hospital for Special Surgery

•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.
Chemical Photography of Bone Composition
1a
PMMA
PO
4
3-
AMIDE I
AMIDE II
1700
1600
1500
CO3=
1400
Wavenumber
1b
1300
1200
1100
1000
(cm-1)
5.000
1c
4.000
3.000
2.000
1.000
0
Mineral
Mineral:Matrix
3
Wt % CO
0.41
11.6
0.39
0.37
10.4
0.35
0.33
9.2
0.31
0.29
8.1
0.27
0.25
7.0
0.23
0.21
5.8
0.19
0.17
4.7
0.15
1d
1.000
0.8000
0.6000
0.4000
0.2000
0
Crystallinity
Carbonate
2-
Chemical Photography of Bone Composition
1a
PMMA
PO
4
3-
AMIDE I
AMIDE II
Sugars
Lipids
1700
CO3=
1600
1500
1400
Wavenumber
1b
1300
1200
1100
1000
(cm-1)
5.000
1c
4.000
3.000
2.000
1.000
0
Mineral
Mineral:Matrix
3
Wt % CO
0.41
11.6
0.39
0.37
10.4
0.35
0.33
9.2
0.31
0.29
8.1
0.27
0.25
7.0
0.23
0.21
5.8
0.19
0.17
4.7
0.15
1d
1.000
0.8000
0.6000
0.4000
0.2000
0
Crystallinity
Carbonate
2-
First Report 1998
Cell 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.
Abstract
A 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 mineralto-matrix ratio and mineral maturity as a function of distance
from an osteon.
Validated FTIRI Parameters
Mineral:Matrix Ratio is Linearly Related to
FTIR mineral:matrix
ratio(Mineral
correlates
with ash fraction
Ash Weight
Content)
1.00
Ash Fraction
Fractional Ash Weight

r2 = 0.83
p < 0.05
0.80
0.60
0.40
0.20
0.00
0
10
20
30
40
50
Spectroscopic Mineral:Matrix Ratio
FTIR
Mineral:Matrix Ratio
60
assessed by x-ray diffraction
180
dimension
c-axis
c-Axis
Thickness(Ǻ)
(A)

1030:1020 Peak Area Ratio Reflects
FTIR crystallinity correlates
with c-axis length
Crystal Size
170
R2=0.98
160
150
140
130
0.50
1.00
1.50
2.00
1030/1020
Area Ratio
FTIR
Crystallinity
2.50
3.00
Carbonate:Phosphate
Ratio Validated Chemically
Magne et al., Bone, 2001
Collagen Maturity (XLR)
Whole bone Chemistry
Bone surface FTIRI
Cortical Periosteal surface
Paschalis et al., Bone, 2011
FTIRI Parameters
Correlate with Mechanical
Properties
Vertebral bone strength correlates with
vertebral crystallinity
Stress (MPa)
30
25
20
R2= 0.29 p= 0.03
15
0.6
0.8
1.0
1.2
1.4
1.6
1.8
1030/1020 (area ratio)
Weinstein et al., Aging Cell, 2010
2.0
2.2
Judex et al, Calcif Tissue
Int, 2005
Bone Volume 47, Issue 6 2010 1030 - 1038



FTIRI is a transmission experiment = thin
sections
Imaging Spectrometer
Software for data processing
 Embedding media: PMMA, Spurs, LR-white
 Zebra fish and calvaria, cell culture
Scales
D16, 4P
EFFECT of EMBEDDING MEDIA
PMMA, Spurr, but not GMA can be removed by
spectral subtraction.
Boskey et al, BBA, 2006
Tissue Heterogeneity:
Select a Single Tissue for Comparison
Values normalized to those at
the iliac crest. Missing bars
indicate that the tissue type
was not present in the biopsy,
n=4
•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.
(Donnelly et al., JOR, 2011)



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
Table I: Infrared Parameters used in Imaging of Bone
-1
Parameter
Wavelength Range (cm )
Mineral:Matrix
900-1200/1585-1720
Carbonate:Phosphate
850-890/900-1200
Collagen Maturity
1660/1690
Crystallinity
HPO4 substitution
1030/1020
1128/1096
Validation
6
Ash Wt.
Chemical
15
analysis
Chemical
16
analysis
6
X-RD
Model
17
compounds
Correlates with
BMD and ash weight, fracture
18,19
risk
20
Age (increase)
Pyridinoline/deoxypyridinoline;
19
fracture risk
21
Bone strength, fracture risk
17
Bone age (inverse)
References available on request
Applications : Answering
Specific Questions about
Bone
5
7
10
6.5
1.35
1.3
1.35
20 25
Pixels
30
1.3
1.35
1.2
10
15
35
5
20
10
5
10
15
20 25
Pixels
30
1.25
1.3
1.35
1.2
1.25
5
15
35
20
5
10
15
20 25
Pixels
30
1.3
10
1.35
1.2
1.25
5
15
35
20
5
10
15
20 25
Pixels
30
1.25
15
35
1.2
20
100%
5
50%
6
1.3
1.2
10
10
15
20 25
Pixels
30
35
10%
Pixels
15
Pixels
10
Pixels
5
Pixels
20
15
1.25
5
15
Pixels
Pixels
5
10
20
5.5
Mineral
to
25
Matrix
30
Ratio
5
4.5
4
35
SHG
40
10
20
Pixels
30
Gourioon-Arsiquaud et al JBMR, 2010
10%
20%
30%
40%
50%
60%
70%
80%
90%
6
Min/Matrix ratio
5.5
5
4.5
2
R = 0.8969
4
2
R = 0.8328
2
R = 0.8566
3.5
0
5
10
15
20
Baboon Age (years)
25
30
100%
2.5
Interstitial tissue has higher Min/Mat
than secondary or old osteons
M in er al/ M at r ix
7
FTIR
6
Secondary
Interstitial Osteon
5
4
Tissue
3
2
Old Osteon
1
0
Se c ond
Cadaveric Femurs
Cadaveric Femurs
Male
(n = 12)
Middle-aged
(n = 6)
Old
(n = 6)
O
o s lt d
e o O Is n
t te e
o rn s t i t i a l
Se c ond
Oo sl t de oO Is n
t te e
o rn s t i t i a l
M id dle Aged
Secondary Osteons x 3
Old Osteons x 3
Interstitial x 3
M. Reyes, Dissertation, U Tx San Antonio


Collagen Maturity
Crystallinity
[Gourion-Arsiquaud et al. 2009]
Crystallinity
* p<0.05; **p<0.001;
n=52
Fracture (0/1) = bo +b1BMD +b2age +b3 Rx +b4(min/mat) +b5(xstl) + b6(XLR) + b7(CO3/P)
Studies of KO animals: DMP-1 KO
Min//Mat
XST
Ling et al., JBMR 2005
8
4
0
fro/fro
fro/fro oim/oim -/Mineral/Matrix
fro/fro
-/-
12 Mineral/Matrix
12
8
WT
8
+/+
1.5
0
1
0
im
Carb/Phosphate
-/+/+
m
fro/fro
0.5
-/+/+
0.008
0.004
1.5
1.5
0
11
fro/fro
oim/oim
fro/fro
fro/fro
+/+
Carb/Phosphate
-/-+/+
Crystallinity
4*
4
-/-
+/+
WT
WT
-/+/+
0.008
oim/oim 0.004
oim/oim
0
Crystallinity
-/- -/Crystallinity
fro/fro oim/oim
+/++/+
*
Carb/Phosphate-/Carb/Phosphate
0
0.0080.008
fro/fro
oim/oim
+/+
0.0040.004
0.5
0.5
00
0
fro/fro
fro/fro oim/oim
oim/oim
0
Coleman, et al. Bone,
fro/frooim/oim
oim/oim
2012 fro/fro
-/
+/
Effects of Treatment
Placebo
ALN
Parameter Mean
77
66
*
*
55
44
33
2
1
0
*
2
*
1
0
M/M
C/P
C/Am I
Xst
Cortical Bone
XLR
M/M
C/P
C/Am I
Xst
XLR
Cancellous Bone
Mineral/Matrix significantly increased in women
without fractures treated 2 yrs with ALN

Healthy bone has a broad distribution of tissue
properties reflecting the presence of new and old
bone
A)
350
300
Numbers of Pixels
250
200
FWHM
150
100
50
0

6.5
6
5.5
5
4.5
A heterogeneous tissue may be better able to resist
crack propagation [Burr & Martin 1986]
Heterogeneity is Decreased in the ALN treated patients
Placebo
ALN
6
5
FWHM
4
*
*
3
2
*
*
*
*
*
*
1
0
M/M
C/P C/Am I XLR
Cortical
XST
M/M
C/P C/Am I XLR
Cancellous
XST
Factor Analysis
F1
Factor Loadings
F4
F6
F2
F5
0.998
micrometers

20
0.996
40
0.994
60
0.992
0.99
80
0.988
100
0.986
F6
F3
120
140
20
40
60
80
100
micrometers
120
F1
0.984
0.982
140
160
Spevak et al., Calcif Tissue 2013
1100
1000

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

nanoIR Spectra Collected at Different Distances from Osteon Center
AFM image 60 x 5 μm
35
30
15μm
25
40μm
59μm
20
15
10
5
0
1800
1600
1400
1200
1000
800
© 2012 Anasys Instruments