Mapping the Organic and Inorganic Components of Bone
C. J. Buckley1, N. Khaleque1, S. J. Bellamy1, M. Robins2, X. Zhang3
1
Department of Physics, King’s College London, Strand, London WC2R 2LS, UK
2
Department of Physiology, King’s College London, Strand,
London WC2R 2LS, UK
3
Department of Physics, State University of New York,
Stony Brook, NY 11794, USA
Abstract. A mapping technique which uses a scanning transmission soft X-ray
microscope (STXM) is described. The technique has been developed and used
to quantitatively map the calcium mineral and protein mass thicknesses in
undemineralised, unstained, thin bone sections. Near complete femoral-neck
sections of sibling normal and ovariectomised mice have been mapped. The
results show the quantitative relationship between calcium and protein on the
macro and microscopic scales for both tissues.
1 Introduction
A number of methods exists for measuring and mapping bone mass [1]. On the
macroscopic and cellular scale, electron probe microanalysis (EPMA) [2], electron
energy loss spectroscopy (EELS) [3], light [4] and infrared [5] microscopies are available. While these techniques have a number of positive attributes, they are not well
suited to the quantitative mapping of mineralised tissue due to either very long pixel
times, or specimen damage. EPMA is primarily sensitive to elemental composition,
whereas EELS and IR imaging offer chemical state contrast. In the case of EELS, its
use is generally limited to inorganic atoms and molecules as significant specimen
damage usually results with organic specimens. IR micro-analysis does not produce
significant specimen damage, but it is impractically slow in imaging mode, and has
poor spatial resolution.
With the advent of bright X-ray sources and improved optics, microscopy
with energy-tunable X-ray probes [6] has opened up some remarkable mapping
possibilities on mineralised tissues [7, 8]. Low energy, mono-energetic X-rays can be
used in transmission mode to quantitatively map both the distribution of the organic
and inorganic molecules. This is achieved by utilising the absorption differences
obtained via spectral features at the carbon and calcium K and L edges respectively.
In the study of osteoporotic bone, it is the distribution and relative amounts of calcium
based mineral and collagen (protein) which are of interest, as collagen forms the
template for mineralisation. In the study reported here, we have quantitatively mapped
and compared the calcium and protein content of femoral neck section in normal and
ovariectomised mice. The purpose of ovariectomisation was to produce a mouse with
low oestrogen levels and cause an early onset of osteoporosis.
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C. J. Buckley et al.
2 Materials and Methods
One of a pair of female sibling mice was ovariectomised at 100 days old to suppress
the production of oestrogen, and the pair were sacrificed at 209 days. The mouse
femurs were removed and fixed in formal saline for ten days and dehydrated by
graded ethanol solutions at room temperature. These were then impregnated with LR
White methacrylate resin for a period of four weeks at 4o C, after which they were
cured at 60oC for 18 hours. The blocks were trimmed to the femoral neck surface, and
cut to the centre of the neck. Sections were cut using a microtome equipped with a
diamond knife set at an angle of 4o to a thickness of 200 to 400 nm. The whole-neck
sections were transferred to a silicon nitride support membrane and imaged in the
STXM.
3 Scanning Transmission X-Ray Microscopy
The STXM at Brookhaven National Laboratory [9] was used for the work reported
here. The undulator source and spherical-grating monochromator provided monoenergetic photons for the microscope. A zone plate X-ray lens demagnified the monoenergetic source forming an X-ray probe of 50 nm in diameter. The specimen was
scanned, and the transmitted intensity was detected by a gas-flow X-ray counter. The
spatial resolution was controlled by the step size of the sample stages, and the defocus
of the X-ray beam. The pixel times for the images shown here varied from 5 ms to
40 ms. The transmission (τ) of the mono-energetic X-rays by a given point in a
sample composed of n elements, having a thickness t, is given by equation (1),
where µi is the mass absorption constant of the ith element, and ρi its density. Note
that ρit is the mass of the element per unit area sampled by the X-ray probe and is
referred to as the mass thickness.
n
τ = exp( − ∑ µ i ρi t)
(1)
i =1
4 Calculation of the Calcium Maps
The creation of quantitative calcium maps is based on the technique of absorption
differences between images formed using a number of X-ray energies close to the
calcium L absorption edge. An absorption spectrum is shown in Fig. 1. In its simplest
form, the technique uses two images either side of the calcium L absorption edge, and
the mass thickness of calcium is found by subtracting the optical densities of these
two images and dividing through by the difference in absorption coefficients, i.e.
ln( τ 1 ) − ln( τ 2 )
[ρt]Ca ≈
(2)
µ 2 − µ1
where the subscripts refer to the two energies used.
A more accurate measurement of the calcium mass thickness is obtained if the
difference in absorption due to other elements in the specimen at the pre and post
calcium L edge energies is taken into account. The absorption changes produced by
Mapping the Organic and Inorganic Components of Bone
II - 49
the calcium-salt mineral elements can be accounted for by calculation, however the
changes associated with the organic matrix can only be determined by mapping the
carbon mass thickness ([ρt]C) at the carbon K-edge. The details of how this is
achieved has been reported [10], and the mass thickness of the calcium is calculated
from
ln( I 1 I 02 / I 2 I 01 ) − (µ C 2 − µ C1 )[ρt ] C
(3)
[ρt ]Ca =
(µ Ca 2 − µ Ca1 ) + ∑ mj=1 [µ mj2 − µ mj1 ]k j
where µC1 and µC2 are the mass absorption coefficients of carbon at the pre- and post
calcium L edge energies, µmj2 and µmj1 are those for the elements associated with the
mineral at the pre- and post calcium L edge energies, while kj is the fractional density
of the jth element in the mineral with respect to the calcium density.
Fig. 1. An X-ray absorption spectrum
taken through a mineralised area of a
normal bone section. The energies
used for mapping were 342.5, 350.6
and 359.4 eV.
Fig. 2. X-ray absorption spectra taken
though L R White embedding medium
(solid) and BSA protein (dotted). The
energies used for mapping were: 281.8,
292.4, 293.05 and 302.4 eV.
The aim of the work reported here was to map both calcium and the protein in the
mineralised tissue sections. The change in absorption cross-section at the pre and
postedge energies of the calcium L edge are similar in magnitude to those for carbon
at the carbon K edge. However, carbon has a considerably greater atomic abundance
than calcium in the specimens, and the thickness of the specimens needs to be 300nm
or less to provide sufficient transmission in order to map the organic components with
a good signal to noise ratio. In order to obtain sufficient absorption by calcium on this
thickness, the NEXAFS peak at 350.6 eV in figure 1 was used. The method by which
quantitative calcium maps using NEXAFS peaks can be obtained is detailed in
Buckley [10]. The use of the NEXAFS peak in combination with the analysis of
equation (3) results in a high signal to noise quantitative calcium map with an
accuracy limit of about 3% imposed by photon statistics. The lower detection limit is
close to 1x10-7 g/cm2 [9] I.e. for a 200nm thick section this is equivalent to 5 mg/cm3.
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C. J. Buckley et al.
5 Calculation of the Protein Maps
There are two major carbon based components in the sample: the embedding material
and the biochemical organic components. By imaging the specimen at energies below
and above the carbon K absorption edge, the total carbon mass thickness can be
measured . This is similar to the calcium mass thickness measurement described in
the previous section. To map and measure the protein mass thickness alone, the fine
structures in the carbon absorption spectra were also used. These fine structures near
the absorption edge are due to the covalent bonds the carbon makes with adjacent
atoms. The energy position of these peaks is influenced by the different chemical
environment surrounding these bonds. Different organic components of the specimen
can therefore be distinguished using the size and position of these peaks.
The embedding material is primarily bisphenol A dimethyl acrylate and
methyl methacrylate, while the organic component in the tissue is mainly collagen.
Fortunately, the differences in their chemical structures give rise to the slightly
different carbon edge spectra as shown in figure 2. The differences allowed us to
separate collagen from the embedding media by imaging at the energies detailed in
the spectra captions. If the chemical formula of the compound to be mapped is
known, then the absorption coefficients can be obtained from tabulated data [11]
which are valid at energies which are not close to the fine structure. The absorption
coefficients for the NEXAFS peaks can be obtained from spectra which are then
normalised by the cross-edge absorption coefficients obtained from tabulated data.
These coefficients can be combined with the transmission images to obtain
quantitative maps of the principal components. The mineralised tissue sections were
treated as being composed of three components: protein (collagen), embedding
medium and mineral. Protein and embedding medium maps were obtained by using
images of the same area taken at four different energies.
There are a number of possibilities for determining the mass-thicknesses of
the protein and embedding media. These are, direct method (such as for the calcium
map), square matrix inversion [12], and singular value decomposition [13] (SVD).
There are several energies at which a difference is observed in absorption coefficient
between the protein and the embedding medium. To optimise the separation between
the protein and embedding medium, more than two energies were used. An advantage
of SVD is that it can be used on an over-determined data set where there are more
optical density equations than unknown mass thickness values. SVD produces a least
squares fit to the mass thickness values produced by the combination of the overdetermined data. This method was applied to determine the protein and embedding
medium mass thicknesses of the bone sections. The sensitivity of the technique for the
protein on the sections used in this study was about 10-6 g/cm2. which was limited by
photon statistics.
6 Results
The results shown in Fig. 3 are the raw transmission images required to make both the
calcium and the protein maps. The images on the top row of Fig. 3 were formed using
the X-ray energies: 281.8, 302.4, 292.4 and 293.05 eV from left to right. These were used
Mapping the Organic and Inorganic Components of Bone
II - 51
to calculate the embedding medium and protein maps shown in Figs. 4a and 4b. The
images in the bottom row of Fig. 3 were formed using the X-ray energies 342.5,
359.4 and 350.6 eV and were used to make the calcium map of Fig. 4c. The calcium
maps can be used to measure the mineral (calcium hydroxy apatite and calcium
carbonate) mass thickness by multiplying the map values by a factor of 2.5.
Fig. 3. Scanning X-ray transmission images of unstained, undemineralised bone section from
the femoral neck of normal mouse sacrificed at 209 days. The transmission images of the top
row (left to right) were formed using the X-ray energies: 281.8, 302.4, 292.4 and 293.05 eV (at
the Carbon K absorption edge). While those of the bottom row were made at the X-ray
energies: 342.5, 359.4 and 350.6 eV (at the calcium L absorption edge). The set of even images
were used to make quantitative maps of the embedding medium, protein and calcium.
A set of seven images were also taken for each field of view on the ovariectomised
mouse sections. The low magnification maps presented in figure 4 are of the majority
of the femoral necks. Higher resolution maps were made of the top right area of the
ovariectomised (figure 5). These are the calcium and protein maps together with an
embedding medium map which highlights the bone forming cells close to the minerlising cartilage. The mass thickness scales on figures 4 and 5 can be converted to the
concentrations in grams per cubic centimeter by dividing the mass thickness value by
the section thickness. The thicknesses of the sections from the normal mouse and
ovariectomised mice were 205 nm and 375 nm respectively.
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C. J. Buckley et al.
40
4a
80
4d
35
4b
35
4e
8
4c
12
4f
Fig. 4. Maps of embedding medium (a & d), protein (b & e) and calcium (c & f) of unstained,
undemineralised sections of normal (left) and ovariectomised (right) mouse femoral neck. The
mass thickness is indicated by an increasing brightness scales, and the maximum values are
indicated in units of g/cm2. The scale bars are equivalent to 100 µm.
Mapping the Organic and Inorganic Components of Bone
38
II - 53
Fig. 5a. Embedding medium map of
the top right area indicated on figure
4b. This is the area between the cortical
bone and the mineralising cartilage.
The embedding medium substitutes hydrated volume, and provides a useful
means of highlighting cells in relation
to the organic and mineralising matrix.
The scale bar represents 5 µm and the
brightness scales are indicated in units
of g/cm2.
25
Fig. 5b. Protein map. The high concentration of protein surrounding the
cells is the collagen matrix exuded by
the cells. The collagen forms the template for mineralisation. The scale bar
represents 5 µm and the brightness scales are indicated in units of g/cm2.
12
Fig. 5c. Calcium map. The more
mature collagen matrix is on the upper
right and lower left, and shows considerable mineralisation. Initial mineralisation islands can be seen in the vicinity of the cells. The structure is typical
of mineralising cartilage. The scale bar
represents 5 µm and the brightness scales are indicated in units of g/cm2.
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C. J. Buckley et al.
7 Discussion
The low resolution images of Figs. 4 show embedding medium, protein and calcium
maps of near complete sections of the majority of the femoral neck at equivalent neck
positions in the normal (4a, 4b & 4c) and ovariectomised (4d, 4e & 4f) mouse bone
sections. Inspection of these maps show the enlarged marrow cavities in the
ovariectomised mice. Further, the density of the protein is seen to increase with
radius in the normal bone, but not so in the ovariectomised bone. The increase in
protein concentration with radius in the normal bone is indicative of the manufacture
of new bone, while the relative uniformity of the protein in the ovariectomised bone
suggests a less active remodeling caused by the hormone imbalance in the animal.
The ovariectomised sample had a section thickness of nearly double that of
the normal mouse sample. This can clearly be seen by inspection of the mass
thickness values in the embedding medium periphery of maps 4a and 4d, where 4b
shows values which are almost double those of 4a. However, inspection of the protein
mass thickness values of figures 4b and 4e show similar maximum values. This
means that the density of the protein in the mineralised areas is considerably lower in
the ovariectomised sample than in the normal sample. An appreciation of the reduced
protein concentration can be gained by comparing the mass thickness values of the
protein in the haemopoietic tissue in the cavities with those in the mineralised areas.
The protein mass thickness in the mineralised areas is considerably greater than that
in the haemopoietic tissue in the normal sample. While the same comparison for the
ovarietomised sample shows a smaller difference. Also, the ovariectomised sample
shows a higher concentration of embedding medium in the mineralised areas, which
indicates a greater micro-porosity in these areas. These findings challenge what has
been the prevailing view that the matrix of osteoporotic-like bone always shows a
normal composition, and support the findings of others (e.g. Diebold et. al. [14]) who
observe significant differences in composition between normal and osteoporotic bone.
To demonstrate the ability of the technique to make quantitative maps in
regions of cellular activity, the top right corner of the ovariectomised sample (marked
with a rectangle in figure 4d) was mapped. The results are shown in figure 5 where
the embedding medium map is shown together with the calcium and protein maps.
The embedding medium (figure 5a) primarily fills the hydrated cavities, highlighting
the matrix-forming cells. Figure 5b shows the higher concentration of protein
(collagen) matrix around the cells while figure 5c shows the calcium distribution in
the mineralising matrix. The calcium mass thickness is greater in the more mature
matrix in the top right and lower left regions.
This initial study using the technique demonstrates the ability of scanning
transmission X-ray microscopy to quantitatively map organic and inorganic constituents of bone over a considerable spatial dynamic range without stain or demineralisation. The analysis presented here has concentrated on calcium, protein and embedding medium. However, it should also be possible to employ the technique to map
other components such as calcium carbonate in these specimens.
Mapping the Organic and Inorganic Components of Bone
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8 Future Work
Bone tissue contains several types of collagen. It has been recognised that some types
of collagen are readily mineralised, while others are not. Future work will map and
measure the protein and mineral densities on animals which have genetically induced
deficiencies of specific types of collagen. The data will be combined with that from
other forms of microscopy on the same samples to gain an insight into the
mechanisms of mineralisation, its suppression and promotion.
Acknowledgements
The authors would like thank the Stony Brook X-Ray Group for the use of their
microscopy equipment on the X1a beamline of the NSLS at Brookhaven National
Lab.
The zone plates used in this work were provided by Erik Anderson of the Centre for
X-Ray Optics, and the work was supported in part by a grant from the NSF grant
number BIR-9316594 and from funds provided by the department of physics at KCL.
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