Full text of DOI:10.1359/jbmr.2001.16.5.893

JOURNAL OF BONE AND MINERAL RESEARCH
Volume 16, Number 5, 2001
© 2001 American Society for Bone and Mineral Research
Infrared Microscopic Imaging of Bone: Spatial
Distribution of CO32Ϫ
H. OU-YANG,1 E.P. PASCHALIS,2 W.E. MAYO,3 A.L. BOSKEY,2 and R. MENDELSOHN1
ABSTRACT
This article describes a novel technology for quantitative determination of the spatial distribution of CO32؊
substitution in bone mineral using infrared (IR) imaging at ϳ6 ␮m spatial resolution. This novel technology
consists of an IR array detector of 64 ؋ 64 elements mapped to a 400 ␮m ؋ 400 ␮m spot at the focal plane
of an IR microscope. During each scan, a complete IR spectrum is acquired from each element in the array.
The variation of any IR parameter across the array may be mapped. In the current study, a linear relationship
was observed between the band area or the peak height ratio of the CO23؊ v3 contour at 1415 cm؊1 to the PO34؊
v1,v3 contour in a series of synthetic carbonated apatites. The correlation coefficient between the spectroscop-
ically and analytically determined ratios (R2 ؍ 0.989) attests to the practical utility of this IR area ratio for
determination of bone CO23؊ levels. The relationship forms the basis for the determination of CO32؊ in tissue
sections using IR imaging. In four images of trabecular bone the average CO23؊ levels were 5.95 wt% (2298
data points), 6.67% (2040 data points), 6.66% (1176 data points), and 6.73% (2256 data points) with an overall
average of 6.38 ؎ 0.14% (7770 data points). The highest levels of CO23؊ were found at the edge of the
trabeculae and immediately adjacent to the Haversian canal. Examination of parameters derived from the
phosphate v1,v3 contour of the synthetic apatites revealed that the crystallinity/perfection of the hydroxyap-
atite (HA) crystals was diminished as CO23؊ levels increased. The methodology described will permit evalu-
ation of the spatial distribution of CO23؊ levels in diseased and normal mineralized tissues. (J Bone Miner Res
2001;16:893–900)
Key words: carbonate, bone, infrared imaging, infrared microspectroscopy, Fourier transform infrared
in normal bone have been reported for various pathological
conditions.(12–16) The relationship between CO23Ϫ content
and the structure of the mineral phase was investigated as a
basis for understanding age-related alterations.(17) Carbon-
ate substitution affects the mineral solubility(5,8,9) and the
presence of CO23Ϫ is thought to play a significant role in
bone resorption.(18) Studies with point-by-point infrared
(IR) microspectroscopy have shown that qualitative changes
in the carbonate/phosphate ratio can provide insight into
the effects of matrix modification and age in transgenic
INTRODUCTION
HE MINERAL phase in bone consists of poorly crystalline,
carbonate-containing hydroxyapatite (HA). Structurally,
T
replacement of PO43Ϫ in the HA lattice by CO23Ϫ leads to a
change in lattice dimensions and increased disorder.(1–7)
The level of CO23Ϫ in bone mineral varies with the age of the
individual and on average probably exists at ϳ6 wt%(8)
although a range of values has been suggested.(9,10) The
level may depend on anatomical location (e.g., trabecular
vs. cortical).(11) Levels different from the range encountered animals and human bone.(19–21) This work describes the
1Department of Chemistry, Rutgers University, Newark, New Jersey, USA.
2Mineralized Tissues Laboratory, Hospital for Special Surgery, New York, New York, USA.
3Ceramics and Materials Engineering, School of Engineering, Rutgers University, New Brunswick, New Jersey, USA.
893

894
OU-YANG ET AL.
development of a method to determine quantitatively the
spatial distribution of CO23Ϫ in mineralized tissues with IR
imaging and will enhance our understanding of the signif-
icance of carbonate in normal and diseased mineralized
tissues.
MATERIALS AND METHODS
Preparation of carbonated HA
Type B carbonate apatites were prepared following the
procedure of Penel et al.(30) Briefly, a phosphate solution
[0.216 M (NH4)2 HPO4 in 30 ml of H2O and 5 ml of
NH3 ⅐ H2O with varying levels of NaHCO3] was added very
IR spectroscopic investigations of homogenized bones
and teeth(22–24) have shown that CO23Ϫ ions generally sub-
stitute for the two anionic sites in the HA lattice for PO34Ϫ slowly over a 3-h period to a constantly stirred calcium-
containing solution [0.25 M Ca(NO3)2 ⅐ 4H2O dissolved in
30 ml of H2O and 10 ml of NH3 ⅐ H2O] at 80°C. The
precipitates were allowed to mature at 80°C for an addi-
tional 2 h. The precipitates were filtered, washed briefly
with distilled water, and dried at 60°C overnight. All re-
agents were analytical grade. Reaction conditions were kept
constant except for different levels of sodium bicarbonate.
ions (type B carbonate, the major substitution site in bone
and dentin) or for OHϪ ions (type A carbonate, which
occurs at a significant level in dental enamel). In addition to
these insertion sites, a labile carbonate species was identi-
fied and is thought to represent surface carbonate.(7) The
vibrational mode widely used for identification of these
species is the out-of-plane deformation, the v2 mode, at 879
cmϪ1 in the free ion, which undergoes substitution site-
dependent spectral shifts on insertion into the HA lattice.
The spatial distribution of the percent of each type of
carbonate substitution in bone has been reported
elsewhere.(7,8,22–24) Polarized IR studies of the v2 mode also
have provided information about the orientation of the
CO23Ϫ planes in the HA of mineralized turkey tendon.(25)
A limitation of the application of traditional IR spectros-
copy to the study of tissues is the necessity for sample
homogenization before spectral examination. This process
renders it impossible to monitor spatial variations in the
tissue constituents. Yet, spatial variations in these properties
are evidently a major determinant of biological function.
The application of IR microscopy techniques (either using
point-by-point methods or array detector-based microscopic
imaging) serves to overcome the limitations of sample ho-
mogenization. In previous publications(26,27) our laborato-
ries have established the feasibility of acquiring IR micro-
scopic images from normal and abnormal states of
mineralized tissue and cartilage. Several IR spectroscopic
parameters have been developed for molecular character-
ization of the mineral and protein components. Although
some of these parameters that measure the mineral content,
mineral crystallinity, and collagen cross-linking can be ap-
plied in both point-by-point Fourier transform infrared
(FTIR) microspectroscopy and IR imaging,(28,29) the afore-
mentioned carbonate analysis is not applicable because the
v2 mode of CO23Ϫ lies outside the operating range of the
current generation of mercury-cadmium-telluride array de-
tector elements, which have a low frequency cut-off of
ϳ900 cmϪ1. The only other CO23Ϫ mode that is currently
feasible for IR imaging is the v3 mode, which for B type
substituted CO23Ϫ, consists of a spectral doublet with com-
ponents at ϳ1419 cmϪ1 and 1450 cmϪ1. This region of the
spectrum overlaps vibrations from both the protein compo-
nents of the tissue and the embedding material (polymeth-
ylmethacrylate [PMMA]) most frequently used for section-
ing the tissue, thus rendering quantitative determination of
CO23Ϫ somewhat awkward. Nevertheless, the current study
shows the feasibility of using spectral subtraction tech-
niques to overcome spectroscopic interference in this spec-
tral region and therefore to use the v3 mode for quantitative
imaging of CO23Ϫ in bone.
FTIR and X-ray diffraction analysis
Carbonate apatite crystals were analyzed by FTIR as
potassium bromide (KBr) pellets (200:1, wt/wt) on a Matt-
son RS-1 spectrometer (Mattson, Madison, WI, USA) with
512 scans coadded at 4 cmϪ1 resolution. Interferograms
were apodized with a triangular function and Fourier-
transformed with one level of zero filling. The sample
compartment of the spectrometer was purged constantly
with dry air generated from a Whatman gas generator
(Whatman, Haverhill, MA, USA).
X-ray spectra were collected on a Siemens D-5000 Pow-
der Diffractometer with automatic sample feeder (Siemens,
Iselin, NJ, USA) using Ni-filtered Cu-K␣ (1.545 A) radia-
tion. Ground samples were scanned from 24° to 37° (2␪) at
0.05° intervals. Data were accumulated for 4 h to achieve
good signal/noise ratios.
Two-dimensional IR analyses
A detailed description of the two-dimensional (2D) IR
correlation spectroscopy techniques developed by Noda(31)
as applied to the study of HA crystallinity has been pub-
lished elsewhere.(32) The spectra are normalized to the in-
tegrated peak areas to compensate for 2D features arising
simply from differences in sample concentrations rather
than from alterations in spectral contours.
FTIR imaging
Iliac crest biopsy specimens were acquired as part of
routine diagnoses and provided under an Institutional Re-
view Board (IRB)-approved protocol by the Pathology De-
partment of the Hospital for Special Surgery. Samples had
been fixed in ethanol, embedded in PMMA, and were cut
into ϳ5-␮m-thick sections before placement between two
BaF2 windows on the instrument stage. Spectra were ac-
quired with a BioRad Sting-Ray system (BioRad, Cam-
bridge, MA, USA). In this device, a 64 ϫ 64 element
mercury-cadmium-telluride (MCT) focal plane array detec-
tor, mapped to a 400 ␮m ϫ 400 ␮m spot at the focal plane
of an IR microscope, is coupled to a step-scan interferom-
eter. Routinely, data from one scan of 1024 steps with 81

SPATIAL DISTRIBUTION OF CO3
895
FIG. 2. The linear relationship between the analytically determined
weight percent CO23Ϫ and the intensity ratio of CO32Ϫ v3 mode to the
PO34Ϫ v1,v3 mode. The regression equation for this determination is Y ϭ
FIG. 1. A series of mid-IR spectra of HA synthesized with (bottom
to top) increasing levels of CO23Ϫ (wt% in the apatitic phase) as
follows: a, 1.05; b, 3.33; c, 4.35; d, 7.80, e, 8.78. The spectroscopic 6.85 ϫ 10Ϫ3 ϩ 0.0349X, where Y ϭ I1415/1030 and X ϭ weight percent
features of interest in the current work are labeled.
of carbonate determined analytically. The correlation coefficient is
R2 ϭ 0.989.
coadditions at each step were collected at 8 cmϪ1 spectral
resolution with one level of zero filling.
butions at these frequencies in the tissue spectra. For each
spectrum in the IR imaging data sets, these correction fac-
tors were applied globally to the entire spectrum, thereby
minimizing contributions from nonmineral components of
the tissue to the CO23Ϫ v3 region.
Carbonate analysis
The CO23Ϫ contents of the synthesized apatites were ob-
tained by standard carbon-hydrogen-nitrogen elemental
analyses. The content of CO23Ϫ in the synthetic HA samples
was taken as 5ϫ the analytical carbon content.
RESULTS
Determination of CO23Ϫ in synthetic HA
Data analysis
A series of FTIR spectra (750–1500 cmϪ1) of synthetic
HA with increasing amounts of CO23Ϫ is shown in Fig. 1. As
the levels of CO23Ϫ increase, several spectral changes are
evident. First, the v2 band near 875 cmϪ1, known(18–20) to
be comprised of at least three underlying subbands corre-
sponding to A-type CO23Ϫ at 878 cmϪ1, B-type CO23Ϫ at 871
The coherent crystal lattice dimension in the c-axis direc-
tion (c-axis particle size and perfection) of synthetic apatites
was calculated from the Scherrer formula:
*
D ϭ K␭/͑B hkl cos␪ ͒,
hkl
where Bhkl is the half-width of the hkl diffraction peak, ␭ is cmϪ1, and relatively minor nonspecific or surface CO32Ϫ at
the wavelength of the radiation, K is a constant assumed 866 cmϪ1 gains in intensity relative to the PO43Ϫ v1,v3
hkl
here to be 0.9, and ␪ is the value in degrees (2␪) of the contour between 950 and 1200 cmϪ1. Second, a similar
diffraction maxima for the hkl reflection.
relative intensity increase is observed in the COϭ3 v3 spectral
IR imaging data were compiled from the 4096 single region. The latter is comprised of a single band near 1419
spectra, which were baseline-corrected in the region of cmϪ1 in the isolated ion (D3h symmetry), but develops a
interest before any spectral manipulations. For the quanti- broad high-frequency shoulder near 1450 cmϪ1 on insertion
tative determination of CO23Ϫ, the contributions of embed- into the HA lattice. The observed ratio of the 1419–1450
ding material (PMMA) and organic matrix (collagen) to the bands is in good accord with the IR data of Rey et al.(7)
original spectra were subtracted based on spectra of the pure for B type CO23Ϫ substitution. This point is discussed in the
compounds. Thus, the intensity of the C A O stretching following paragraph. Finally, increasing levels of CO23Ϫ are
mode of PMMA at ϳ1720 cmϪ1 and the amide I frequency accompanied by broadening of the spectral features within
at ϳ1650 cmϪ1 (for collagen) were used as standards for the phosphate v1,v3 contour, which are indicative of de-
spectral subtraction because there were no mineral contri- creased mineral crystallinity/perfection.

896
OU-YANG ET AL.
FIG. 3. 2D IR synchronous plot for the series of synthetic samples
with increasing levels of CO23Ϫ ion. The solid lines are positive corre-
lation contours. The dashed lines are negative correlation contours.
FIG. 5. Calculated c-axis particle size/perfection as a function of
CO23Ϫ concentration determined from the Scherrer equation applied to
the X-ray powder pattern line widths for the 002 reflection.
It is noted that comparisons of parameters arising from
single bands (peak heights, areas, etc.) are subject to uncer-
tainties arising from thickness variations between samples.
Ratios of areas or peak heights therefore are used to control
for sample-to-sample variations in the weights of the HA
phase used in the analysis. In addition, the use of parameters
involving ratios in IR microscopy compensates for sample-
to-sample thickness variations in microtomed sections. The
drawback of the current measurement is the inherent as-
sumption that the molar integrated area of the phosphate
v1,v3 contour does not change as the mineral crystallinity/
perfection is altered. This conjecture cannot be tested easily
so that the aforementioned intensity ratio must be consid-
ered as an approximation to the relative carbonate concen-
tration. However, the high value of the correlation coeffi-
cient between the spectroscopically and analytically
determined ratios in Fig. 2 (R2 ϭ 0.989) attests to the
practical utility of the current intensity ratio for IR deter-
mination of bone carbonate levels.
Effect of CO23Ϫ on HA crystallinity/maturity
As is evident from Figs. 1 and 2, increasing levels of
carbonate cause substantial alterations in the PO34Ϫ v1,v3
contour. Shape changes in this band are caused by alter-
ations in the positions and widths of the underlying sub-
bands and reflect alterations in mineral crystallinity. Several
standard data reduction protocols including curve-fitting,
derivative spectroscopy, and Fourier self-deconvolution
FIG. 4. X-ray powder diffraction patterns for a series of samples of
HA synthesized with (bottom to top) increasing levels of CO23Ϫ (wt%
in the apatitic phase) as follows: a, 1.05; b, 3.33; c, 4.35; d, 7.80, e,
8.78.
The quantitative relationship between IR spectral param- have been previously used to analyze quantitatively changes
eters and the analytically determined carbonate levels in in this contour when sample crystallinity/perfection is al-
these synthetic samples is shown in Fig. 2. The most useful tered. As discussed elsewhere,(32) each of these methods
IR parameter for eventual imaging applications is the band offers advantages and disadvantages for the analysis of
area ratio of the CO23Ϫ v3 contour to the PO34Ϫ v1,v3 contour. complex contours. Recently, we have shown that the reso-

SPATIAL DISTRIBUTION OF CO3
897
FIG. 6. (A) A series of IR spectra selected from imaging data across trabecular bone showing contributions from PMMA at 1720 cmϪ1 and from
the protein amide I mode near 1660 cmϪ1. (B) The same series of IR spectra as in panel A in which the contributions from PMMA at 1720 cmϪ1
have been removed mathematically while the protein amide I mode near 1660 cmϪ1 are still present. (C) The same series of IR spectra as in panel
B from which the protein amide I mode near 1660 cmϪ1 have been removed mathematically.
lution enhancement techniques of 2D IR spectroscopy can vibrational modes of both the protein component of the
be applied to this contour to examine the subtle changes
induced by alterations in crystallinity/perfection.(32) Figure
3 shows the 2D synchronous plot generated from the set of
samples with increasing levels of CO23Ϫ in the spectral range
of 900-1500 cmϪ1. The two components of the CO32Ϫ v3
contour are correlated negatively with the 1030-cmϪ1 com-
ponent of the phosphate v1,v3 contour. The latter band is
known to increase in intensity as mineral crystallinity/
perfection is enhanced. In previous studies,(33) we showed
that the 1045-cmϪ1 band reflects the amount of B-type
CO23Ϫ. In the current work, a small peak in the 2D synchro-
nous plot is visible at this position, although the bulk of the
change in the contour arises from variation of the 1030-
cmϪ1 component.
tissue and the PMMA embedding material. To compensate
for contributions from these constituents to the CO23Ϫ v3
mode, a double subtraction protocol was used. The success
of this procedure is shown in Fig. 6. The original baseline-
corrected spectra from sites across trabecular bone (i.e.,
varying from low to high levels of mineral) selected from IR
imaging data are shown in Fig. 6A. The same set of spectra
following the scaled subtraction process to eliminate inter-
ference from PMMA is displayed in Fig. 6B. As evident in
Fig. 6B, the subtraction process occasionally results in a
small residual derivative-like feature, arising from slight
spectral shifts in the PMMA C A O stretching band. The
contribution of protein to the 1419 cmϪ1 band was esti-
mated from the ratio of the amide I intensity to the 1419
feature in collagen spectra, which was used as a standard for
subtraction. The resultant set of spectra is shown in Fig. 6C.
The double subtraction process evidently is reasonable,
because it results in CO23Ϫ v3 contours that closely resemble
those in the series of synthetic HA derivatives (compare Fig.
6C with Fig. 1). To illustrate the quantitative determination
of relative CO23Ϫ levels in mineralized tissue as determined
from IR microscopic imaging, the results of analyses of four
trabecular regions are reported. In addition, a series of 36
spectra were selected from IR imaging data across a region
from one of the trabecular bone samples. The spatial vari-
ation in the carbonate/phosphate ratio across trabecular
bone as monitored by the intensity ratio of the CO32Ϫ
v3/PO34Ϫ v1,v3 bands following the double subtraction pro-
cess is shown in Fig. 7A. The highest relative levels of
CO23Ϫ-induced changes in HA crystallinity for this set of
synthetic samples also were evaluated with X-ray diffrac-
tion. A series of X-ray powder patterns (2␪ from 24° to 37°)
of HA with increasing levels of CO23Ϫ is shown in Fig. 4.
There is substantial carbonate-induced line broadening in all
the reflections, but because of limitations with sample vol-
ume, calculations of changes in the a-axis dimension were
not conclusive. The calculated crystal particle size and
perfection derived from the Scherrer equation is plotted for
the 002 reflection in Fig. 5 as a function of the level of
CO23Ϫ substitution. Similar trends were noted for the 300
reflection.
Applications to IR microscopy
The primary difficulty with the use of the CO23Ϫ v3 mode
for IR imaging is the potential interference from overlapped carbonate occur at the edges, with the lowest relative levels

898
OU-YANG ET AL.
FIG. 7. (A) Variation in the intensity ratio of
CO23Ϫ v3 mode to the PO43Ϫ v1,v3 mode across a
trabecular bone sample from a human iliac crest
biopsy specimen. The spectra from which these
data were obtained were selected from the set of
IR imaging data shown in Fig. 6. (B) Variation
in the intensity ratio of two peaks at 1030 cmϪ1
and 1020 cmϪ1 within the PO43Ϫ v1,v3 contour
across the trabecular bone sample shown in
panel A.
evident at the center of the tissue. The variation in mineral 7.4%, the average value determined here lies well within the
crystallinity/perfection as monitored from the intensity ratio
(I 1030/I 1020) is plotted in Fig. 7B and shows the trends in
the direction opposite to those seen in the carbonate/
phosphate levels. The I 1030/I 1020 index is highest at the
center of the bone and decreases toward the edges.
currently accepted range.
The effect of CO23Ϫ substitution on HA crystallinity as
reported previously(5,9,34) also is evident from the 2D IR
data shown in Fig. 3. The 2D IR synchronous plots show a
strong negative correlation between the 1030-cmϪ1 compo-
nent of the PO34Ϫ v1,v3 contour and both components of the
CO23Ϫ v3 mode. In the current instance the correlation is
strengthened by the X-ray diffraction data from the syn-
thetic apatites (Fig. 4). These results are in accord with our
previous investigations that show the 1030-cmϪ1 compo-
nent to be enhanced as the HA crystallinity/perfection is
increased. Increased levels of carbonate thus are indicative
of an HA phase with reduced crystallinity/perfection.
The determination of CO23Ϫ in homogenized bone by IR
is well documented. Some 35–40 years ago, Emerson and
Fischer(35) and Baxter et al.(36) observed a doubling of the v3
mode. They attributed the additional spectral feature near
1450 cmϪ1 to symmetry lowering of the ion in the tissue.
The use of the low-frequency component of v3 to determine
CO23Ϫ in apatites was shown by Featherstone et al.(37) They
determined that the absorbance ratio of this feature to the
phosphate v4 band at 575 cmϪ1 allows an estimate of
carbonate levels to better than Ϯ10% in the biologically
relevant range of 1–12 wt%. The phosphate v4 band is
precluded from the current study because of detector limi-
tations but the v1,v3 mode used instead offers the advantage
of being the strongest band in the IR spectra of mineralized
tissues.
Finally, the variation in relative CO23Ϫ levels in one of
400 ␮m ϫ 400 ␮m sections of trabecular bone as deter-
mined from an IR imaging data set is depicted in Fig. 8A.
The area ratio of the CO23Ϫ v3/PO43Ϫ v1,v3 bands is converted
to weight percent CO23Ϫ in the mineral phase according the
regression equation generated from the data in Fig. 2, color
coded, and plotted as an image. In four images of trabecular
bone the average CO23Ϫ levels were 5.95 wt % (2298 data
points), 6.67% (2040 data points), 6.66% (1176 data
points), and 6.73% (2256 data points). The average value of
CO23Ϫ from these images is ϳ6.38 Ϯ 0.14 wt% (7770 data
points from the four trabecular bones). In one image of
cortical bone from human iliac crest biopsy specimens the
CO23Ϫ levels were 6.40% (3792 data points). The highest
levels of CO23Ϫ were found at the edge of the trabeculae.
The highest relative levels of CO23Ϫ, approaching 10–11%,
occur at in a very small number (Ͻ1%) of pixels near the
edges of the bone. Such high levels of carbonate were seen
only in this trabecular bone sample. In the other samples
studied, the highest weight percent CO23Ϫ observed was 8%
or 9%. A histogram depicting the overall CO23Ϫ distribution
from the four trabecular bone samples are shown in Fig. 8B.
An important issue in the current experiments is the
substitution pattern of CO23Ϫ. Rey et al.(7,22,23) have made a
thorough study of the effect of the substitution type on the
CO23Ϫ v3 spectral region. Pure type A CO23Ϫ in synthetic
samples exhibited a well-defined doublet in synthetic sam-
ples with components at 1463 cmϪ1 and 1435 cmϪ1, a
pattern not observed in the current bone samples. It is
therefore concluded that there is no significant type A
substitution in our samples. Type B CO23Ϫ also presents a
well-defined doublet in synthetic samples with components
observed by Rey et al.(7) at 1456 and 1422 cmϪ1 with a peak
height ratio (calculated from Fig. 3B of Rey et al.(7)) of 1.21.
In our synthetic samples, we have observed the same dou-
DISCUSSION
The current work describes the first quantitative determi-
nation of the spatial variation of CO23Ϫ in mineralized thin
tissue sections using IR imaging. Such a characterization
may lead to improved understanding of the functional role
of the ion in normal and diseased states. It can be used to
correlate variations in microarchitecture with carbonate
level or to examine the relative carbonate content adjacent
to active osteoclasts or quiescent osteoblasts. It also can be
used in tooth sections to distinguish enamel and dentin. The
values of the intensity ratio across trabecular bone reveal an
average value of 6.38 Ϯ 0.14 wt% CO23Ϫ in the apatitic
phase. As reported values of bone CO23Ϫ range from 4 to blet with a linear decrease in the component intensity ratio

SPATIAL DISTRIBUTION OF CO3
899
ACKNOWLEDGMENTS
This work was supported by a Public Health Service
Grant (AR 41325) to A.L.B. and R.M. IR images were
collected in the IR imaging core at the Hospital for Special
Surgery, supported by AR 46121.
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FIG. 8. (A) Variation in the intensity ratio of CO23Ϫ v3 mode to the
PO34Ϫ v1,v3 mode throughout a trabecular bone sample from a human
iliac crest biopsy specimen, acquired from IR microscopic imaging.
The color-coding reflects the actual intensity ratios measured from the
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regression equation from the data in Fig. 2. The number of data points
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array) because the sample does not fill the field of view. (B) The
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from 1.27 to 1.15 as the CO23Ϫ level increased from 0 to
10%. The variation in the ratio may be related to a differ-
ential broadening in the widths of the two components as
the CO23Ϫ level increases. It is evident from a comparison of
Rey’s data(23) with ours that the current bone samples ex-
hibit negligible interference from type A substitution.
Extensions of the current experiment appear feasible. For
example, with appropriate control samples, it should be
possible to image spatial variations in both A- and B-type
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straightforward fashion. In addition, relationships between
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Address reprint requests to:
Dr. Adele L. Boskey
Hospital for Special Surgery
535 East 70th Street
New York, NY 10021, USA
28. Paschalis EP, DiCarlo E, Betts F, Sherman P, Mendelsohn R,
Boskey AL 1996 FTIR microscopic analysis of human os- Received in original form July 21, 2000; in revised form November
teonal bone. Calcif Tissue Int 59:480–487. 15, 2000; accepted December 4, 2000.