Noninvasive Raman spectroscopy of rat
tibiae: approach to in vivo assessment of
Paul I. Okagbare
Steven A. Goldstein
Michael D. Morris
spectroscopy of rat tibiae:
approach to in vivo
assessment of bone quality
Paul I. Okagbare,aDana Begun,bMary Tecklenburg,c
Ayorinde Awonusi,cSteven A. Goldstein,band
Michael D. Morrisa
aUniversity of Michigan, Department of Chemistry, Ann Arbor,
bUniversity of Michigan, Medical School, Orthopedic Research
Laboratories, Department of Orthopedic Surgery, Ann Arbor, Michigan
cCentral Michigan University, Department of Chemistry, Mt. Pleasant,
Abstract. We report on in vivo noninvasive Raman spectro-
scopy of rat tibiae using robust fiber-optic Raman probes
and holders designed for transcutaneous Raman measure-
ments in small animals. The configuration allows placement
of multiple fibers around a rat leg, maintaining contact with
the skin. Bone Raman data are presented for three regions of
the rat tibia diaphysis with different thicknesses of overlying
soft tissue. The ability to perform in vivo noninvasive Raman
measurement and evaluation of subtle changes in bone
composition is demonstrated with rat leg phantoms in
which the tibia has carbonated hydroxylapatite, with differ-
ent carbonate contents. Our data provide proof of the prin-
ciple that small changes in bone composition can be
monitored through soft tissue at anatomical sites of interest
in biomedical studies. © 2012 Society of Photo-Optical Instrumentation
Engineers (SPIE). [DOI: 10.1117/1.JBO.17.9.090502]
Keywords: carbonated hydroxyapatite; fiber-optic probes; in-vivo
Raman; rat tibia.
Paper 12343L received Jun. 11, 2012; revised manuscript received Jul.
29, 2012; accepted for publication Jul. 31, 2012; published online Sep.
Bone undergoes continuous remodeling in response to loading,
local damage, and pathologies. Remodeling of bone changes its
chemical composition, which is an important indicator of its
physiological status and mechanical competence.1,2The ability
to measure changes in chemical composition of bone is, there-
fore, important.2–4Existing noninvasive technologies used to
monitor bone evaluate the morphology and calcium content,
but provide no direct information on other properties of the tis-
sue.5–9Vibrational spectroscopy has proved useful in the study
of bone development, bone biomechanics, and bone pathologies
because it provides this composition information.10,11Unlike
infrared spectroscopy, which provides essentially similar infor-
mation, Raman spectroscopy suffers little from water interfer-
ence; therefore, it is applicable to fresh tissue and in vivo
However, in vivo Raman spectroscopy has its own chal-
lenges, including background fluorescence from tissue compo-
nents (especially skin melanin), interfering protein spectral
bands from overlying soft tissue, and the multiple scattering
that is characteristic of almost all tissues.13These challenges
limit the depth from which Raman signals can be collected.
Realizing the potential for in vivo studies requires fiber-
optic probes that are configured for transcutaneous Raman
measurement on a time scale ranging from a few seconds to
one or 2 min. Our laboratory has developed several fiber-
optic probes based on spatial separation of excitation and col-
lection fibers that allow bone Raman spectroscopy through
layers of overlying tissue.14,15The principle is well known in
tissue spectroscopy; in the literature, it is called spatially offset
Raman spectroscopy.16,17A limitation of spatial offsetting is that
measurement time increases rapidly as the depth of penetration
increases. We have previously demonstrated backscattering in
in vivo collection of murine Raman spectra at depths of a
few millimeters in mice18and low-definition Raman tomogra-
phy in excised tissue at thicknesses of more than 2 cm.19These
measurements haverequired unrealistically long times. Here, we
describe and test a probe configuration adapted from diffuse
optical tomography that reduces measurement time to 60 s at
several positions along a rat tibia.
We have previously reported cadaveric rat tibia studies using
a diffuse tomography-based probe containing 50 collection
fibers terminated in stainless steel ferrules and a single excita-
tion fiber that could be positionedat one ofseveralpointsaround
the tibia.20The fibers were mounted in a conformal silicone
In the present design, the fifty 100-μm silica core fibers are
grouped into 10 bundles of five fibers each, providing greater
mechanical robustness. A single 300-μm silica core excitation
fiber is used. A demountable aluminum fiber holder that can be
positioned reproducibly around the leg of the rat replaces the
silicone holder used previously (Fig. 1).20The fiber holder is
constructed as two pieces to facilitate rapid placement and
removal. O-rings mounted through holes arranged around the
circumference of the holder allow the easy insertion andremoval
of fibers. The design allows a sequence of measurements with
the excitation fiber placed at a different position in each mea-
surement to optimize the collection of bone spectra. The alumi-
num holder is easily positioned at different points along the
tibial diaphysis. The placement of the illumination and collec-
tion probes generates adequate spatial offset for transcutaneous
Raman measurement on rat limbs. The collection fibers are
arranged into a linear array for coupling to the input slit of
a Raman spectrograph. The Raman instrument is an axial
transmissive Raman spectrograph equipped with an 830-nm
excitation laser and a256 × 1024front-illuminated CCD for col-
lection of near-infrared light (RamanRxn1, Kaiser Optical Sys-
tems Inc., Ann Arbor, MI).
Using this probe and a holder with single-fiber illumination,
bone Raman spectra were acquired for 60 s at three regions
along the diaphysis of the tibia of an anesthetized rat,
as shown in Fig. 1(c). This sequence allowed measurement
through different thicknesses of overlying soft tissue. The
acquired Raman data were processed in MATLAB (MathWorks,
Inc., Natick, MA) as previously described to remove cosmic
ray spikes, grating-induced curvature, and grating/detector
Address all correspondence to: Michael D. Morris, University of Michigan,
Department of Chemistry, Ann Arbor, Michigan. Tel: 734-769-3758; Fax: 734-
615-3796; E-mail: email@example.com
0091-3286/2012/$25.00 © 2012 SPIE
Journal of Biomedical Optics090502-1September 2012 • Vol. 17(9)
misalignment.20,21The spectra are reported as wavenumber
shifts and are corrected for detector quantum efficiency variation
after background subtraction
removal).20,22Figure 2(a) presents unsmoothed Raman spectra
from the three diaphysial positions without any removal of over-
lying tissue contributions. As expected, the intensity of the bone
mineral band at 960 cm−1relative to collagen protein bands at
1450 and 1660 cm−1is greater for region 1 (960∕1660 ¼ 3.06),
where the maximum thickness of the overlying soft tissue is
about 1 mm. There are increasing contributions from the
overlying tissue signal in regions 2 and 3 [∼0.5 cm
(960∕1;660 ¼ 1.94) and ∼1 cm (960∕1660 ¼ 1.57) maximum
thickness, respectively]. In every case, one or more detectors
(fibers) views bone underneath a thin layer of tissue due to
the anatomy of the rat leg, so mineral/collagen intensities do not
scale linearly with the maximum soft tissue thickness. Forexam-
ple, in region 3, detector fiber 1 is located over the tibia with
∼5 mm of soft tissue thickness from the underlying bone, while
detectors 2, 3, 4, and 5, and 6, 7, 8, and 9 are more than
∼8.5 mm and ∼14.1 mm, respectively, from the underlying
bone. In region 2, fiber 1 is positioned at ∼3 mm, fibers 2, 3,
4, and 5 at ∼4.6 mm and fibers 6, 7, 8, and 9 at ∼7 mm from the
bone. In region 1, all fibers are positioned with approximately
the same soft tissue separation (∼1 mm) from the underlying
bone. The variation in acquired bone mineral Raman signal
intensities relative to each individual fiber position is shown
in Fig. 2(b) for region 2.
Overlying tissue contributions cause changes in the relative
intensities of the protein bands with respect to the mineral
band. To separate the bone Raman spectrum from soft tissue
contribution, band target entropy minimization (BTEM)23
was performed on the spectra from the three regions using the
first few eigenvectors of the singular value decomposition
(SVD) matrix.18The unsmoothed recovered spectra for the
three regions are shown in Fig. 2(c). Soft tissue contributions
to the spectra are minimized, but not completely removed, after
BTEM and the intensities of the bone mineral band at 960 cm−1
relative to collagen protein bands are enhanced in regions 2 and
3. The incomplete removal of soft tissue by BTEM could be
associated with the short acquisition time, the signal-to-noise
ratio, and the limited number of input spectra into the BTEM
algorithm. These results suggest that this probe configuration
is suitable for longitudinal spectroscopic studies offracture heal-
ing, among other subjects. The probe can be positioned in 1-min
measurements made in 1 min, and the probe removed in a few
seconds. in vivo Raman spectroscopy can consume a small frac-
tion of the 30-min maximum duration of anesthesia.
Carbonate substitution is one of the proposed Raman metrics
for evaluating bone quality.3Because of the proximity of
to measure band intensity accurately.24We demonstrated the
ability to measure small changes in bone compositions using
multilayer phantoms that model geometric, optical, and spectro-
scopic rat leg properties.20,25The phantoms were constructed
with carbonated hydroxyapatite containing different amounts
of carbonate as the bone mineral model.20,25Raman spectra
3ν1to a component of PO−3
4ν3it is notoriously difficult
Fig. 1 Noninvasive Raman spectroscopy of a rat tibia. (a) The probe
holder is fastened to a bed on which the rat lies while anesthetized.
The fibers (D1–D9) are adjustable, as shown with a phantom. (b) The
phantom mimics the anatomy and spectroscopy of rat leg. (c) A rat leg
rendering showing the three regions along the diaphysis where Raman
spectra were obtained.
Fig. 2 Raman spectra from three different regions of rat leg along the tibial diaphysis. Spectra were normalized to the protein band at 1660 cm−1.
(b) Raman spectra from the individual fibers labeled D1–D9 in the wavelength range of 850 to 1100 cm−1. The intensities of the phosphate (bone
mineral) band varied with fiber positions. (c) Recovered bone Raman spectra after BTEM was performed on the data in (a) for the three regions. Soft
tissue contributions are minimized to recover the bone Raman spectrum. Spectra were normalized to the mineral band at 960 cm−1.
Journal of Biomedical Optics090502-2September 2012 • Vol. 17(9)
of the phantoms were acquired at the laser powers and measure- Download full-text
ment times used for the rat tibia. The resulting Raman spectra
are shown in Fig. 3. The band intensities at 1070 cm−1for the
0.3%, 4.7% and 6.9% CO−2
bonate substitution. The width of PO−3
increases with increasing carbonate substitution and shifts to
lower wavenumber (see Fig. 3 insert), as reported for the
salts.24The full-width half-maximum of the PO−3
linearly (r2¼ 0.99) with CO−2
not shown in Fig. 3). These data provide proof of the principle
that small changes in bone composition can be monitored
through at least a 1-cm layer of soft tissue.
Our results complement the results of our previous report on
the distal tibiae of mice, which have a thinner layer of soft
tissue.18Although we have not directly addressed noninvasive
human subject bone spectroscopy, our results suggest that at
some sites, 1-min measurements are feasible.
3values increase with increasing car-
4ν1at ∼960 cm also
content (the linear plot is
The authors acknowledge support of this work through
National Institutes of Health (NIH) Grants R01AR055222
and R01AR056646. The authors also thank Kathleen Sweet
for help with animal handling and Charles Roehm for construc-
tion of the optical fiber holder.
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Fig. 3 Transcutaneous Raman spectra acquired from rat leg. The rat leg
models were constructed using carbonated hydroxyapatite with varied
level of carbonate substitution (0.3%, 4.7%, and 6.9% carbonated
hydroxyapatite) as the tibia model. Differences between specimens
were captured in the Raman spectra. Intensity of the carbonate band
at 1070 cm−1and the width of the phosphate band at 960 cm−1both
increase with carbonate substitution. The expanded view of the spectra
in (a) showing the wavenumber range from 930 to 980 cm−1is pre-
sented as an insert. The position of the PO−3
960 to 958 cm−1with increasing carbonate substitution.
4ν1peak shifted from
Journal of Biomedical Optics090502-3September 2012 • Vol. 17(9)