# Optical Clearing of Cranial Bone

Authors: Elina A. Genina; Alexey N. Bashkatov; Valery V. Tuchin
Journal: Advances in Optical Technologies
 (2008)

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## Abstract

We present experimental results on optical properties of cranial bone controlled by administration of propylene glycol and glycerol. Both transmittance and reflectance spectra of human and porcine cranial bone in vitro were measured. For estimation of absorption and reduced scattering coefficients of the bone, the inverse adding-doubling method was used. The decrease of reflectance of the samples under action of the immersion agents was demonstrated. The experiments have shown that administration of the immersion liquids allows for effective controlling of tissue optical characteristics that makes bone more transparent, thereby increasing the ability of light penetration through the tissue. The presented results can be used in developing of functional imaging techniques, including OCT.


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## Body

## 1. Introduction

Interest
in using optical methods in the development of noninvasive
clinical functional cerebral imaging systems for physiological-condition monitoring [1, 2],  cancer diagnostics, and
therapies [3, 4] increases due to their simplicity, safety,  and low cost in
contrast to conventional X-ray computed tomography, magnetic
resonance imaging, and ultrasound imaging [5–8]. Spectroscopic
techniques are capable of deep imaging of tissues that could provide
information of blood oxygenation and tissue metabolism [1, 2, 9, 10] and detect
brain malignancies [4, 11, 12].
However, the main limitations of the optical imaging techniques, including
diffusion optical tomography, optical coherent tomography, and reflectance
spectroscopy deal with the strong light scattering in superficial tissues,
which cause decrease of spatial resolution, low contrast, and small penetration
depth. One of the prospective solutions of the problem is a reduction of light
scattering of the upper tissue layers that provides improvement of image
quality and precision of spectroscopic information getting from tissue depth
[5, 13–15].It is well known that the major
source of scattering in tissues and cell structures is a refractive index
mismatch between cell organelles, like mitochondria and cytoplasm,
extracellular media and tissue structural components such as collagen and
elastin fibres [5]. The tissue scattering properties can be significantly
changed due to action of immersion liquids [15–20].
Administration of the immersion liquid having a refractive index higher than
that of tissue interstitial fluid and/or hyperosmotic properties induces a
partial replacement of the interstitial fluid by immersion substance and hence
matching of refractive indices of tissue scatterers and the modified
interstitial fluid. The matching, correspondingly, causes the decrease of
scattering. As immersion liquids aqueous solutions of glucose and mannitol,
propylene glycol, glycerol, and other biocompatible chemicals are used [15–20].The possibility of selective translucence of
cranial bone can be very useful in developing techniques of brain functional
imaging. A potential benefit of the optical clearing technique is an
improvement of laser therapeutic techniques that rely on sufficient light
penetration to a target embedded in tissue. Combining optical clearing with
laser radiation could reduce the laser fluence required for a therapeutic
effect. Another application of the optical method is a noninvasive
visualization of brain blood vessels, hematomas, and small pathologic
structures (including cancerous growth) with a high resolution. This is
important for diagnosis and treatment of many diseases such as tumors of brain,
vascular pathologies, and so forth. However, despite the numerous studies of
optical clearing of such tissues as skin, sclera,dura mater, and so forth, the bone optical clearing is not investigated
enough up to now.In
this study, we investigate optical clearing of both human and porcine cranial
bone in vitro under the action of propylene glycol and glycerol.


## 2. Bone Structure and Physical Properties

The structural
components of the bones consist of an inorganic matrix (largely mineralised)
and an organic matrix [21, 22]. The inorganic matrix contains calcium
hydroxyapatite, which is responsible for the compressive strength of bone, and
osteocalcium phosphate. The main components of the organic matrix are collagen,
proteins, blood cells, and lipids [23]. The amount of bone mineral matrix is
16%, the lipid content is 54%, the proteins content is 16%, and water
contributes 16% [23]. It is the
calcium and phosphorus component of the inorganic matrix that makes bone hard
and rigid, and the arrangement of the collagen fibres in the organic matrix
that makes it strong. Porosity of the bones is 5–10% [23].At
microstructural length scales, cortical bone is organised into 200–300 μm diameter secondary osteons [24], which are
composed of large vascular channels (50–90 μm diameter) surrounded by circumferential
lamellar rings (3–7 μm thick), with the so-called “cement
lines” at the outer boundary [25]. At the nanostructural level, the
lamellae are composed of organic type-I mineralised collagen fibres (up to 15 μm in length, 50–70 nm in
diameter, and formed by regular arrangement of subnanostructural collagen
molecules) bound and impregnated with inorganic carbonated apatite nanocrystals
(about 30 nm in length and width, 2-3 nm in
thickness) [26, 27].Water is of significant importance for the living bone and is one of its
major components. Bone water occurs at various locations and in different
binding states. Water in bone may be found associated with the mineral phase,
bound to the organic phase (collagen and cement substance), or free (bulk
water) [28, 29]. The most tightly bound water is the one occupying the calcium
ion coordination sites in the apatite-like crystals (about 35 mg of water/g
mineral) [30]. A significant less tightly bound fraction is water associated
with collagen fibrils. In the unmineralised state as the bone matrix is laid
down by obsteoblasts, the collagen fibers produced contain a large volume
fraction of water (up to 60%) [31]. During calcification, however, apatite
crystals are deposited in the organic matrix, gradually displacing the osteoid
water and reducing it down to a 20% volume fraction. Eventually, this fraction
drops to 10% in senile bone [32]. Bulk water fills the pores of the calcified
matrix, which form a network of interconnecting channels (the lacunocanalicular
system), which communicate the Haversian canals (the bone vascular system) with
the osteocytes, embedded in the mineralized matrix [28, 31]. This communication
network serves for transport of nutrients, waste products, and signaling
molecules from the vascular system to the osteocytes and vice versa. The water channels are
also the transport pathways for calcium and phosphate ions flowing in and out
of bone tissue, which acts as a mineral reservoir for the rest of the organism
[28].Refractive index of the whole
cranial bone has been estimated by Ascenzi and Fabry [33] at various stages of mineralisation to range from
1.555 to 1.564.  Components of the tissue have the following refractive
indices: apatite: > 1.623 [34], hydrated collagen (type I): 1.43 [35], and
lipids ~1.45 [36].


## 3. Materials and Methods

### 3.1. Materials

For this study, five porcine and ten human
cranial bone samples were used. All bone samples were cortical (or compact) bones. The samples of human cranial bone were obtained frompostmortem examinations. All samples were kept in saline at temperature about 5°C until spectroscopic measurements. The bone
samples were measured during 4–6 hours after
autopsy. The area of the samples was about 25×25 mm2. The thickness of each bone
sample was measured with a micrometer in several points over the sample surface
and averaged. Precision of the single measurement was ±50 μm. Thickness of the samples varied from 1.6±0.1 to 5.0±0.5 mm.In this study,
the commercially available propylene glycol (“Reactive,” Russia) and glycerol (“Ph. Eur.,” Germany)
have been used as clearing agents. The refractive index of propylene glycol and
glycerol has been measured by Abbe refractometer at wavelength 589 nm as 1.43
and 1.47, respectively.


### 3.2. Experimental Setups

The measurements of bone reflectance have been
performed in the spectral range 450–1000 nm using a
commercially available optical multichannel spectrometer LESA-5
(“BioSpec,” Moscow,
Russia) with a fiber-optical probe.  The scheme of the
experimental setup is shown in Figure1. The fiber optical probe consists of
seven optical fibers. All fibers had 200 μm core diameter and a numerical aperture of
0.22. The central fiber delivers incident light to the tissue surface, and six
fibers, placed around the central fiber, collect reflected light. Distance
between the delivering and receiving fibers is 290 μm. As a reference, a white slab BaSO4 with a smooth surface has been used. The sample of bone was placed into cuvette
with the immersion liquid. For spectrophotometric measurements, each sample was
removed from the cuvette. Then the sample was again placed in the cuvette.Figure 1
The geometry of the measurements in reflectance
mode with the spectrometer LESA-5:
(1) halogen lamp (2) fiber-optical probe (3) cuvette with a bone sample (4) grating and photodiode array.The total transmittance and diffuse reflectance
measurements have been performed in the 800–2000 nm
wavelength range using the commercially available spectrophotometer CARY-2415
(“Varian,” Australia)
with an integrating sphere (Figure2). Inner diameter of the sphere is 100 mm, size of the entrance
port is 20×20 mm, and diameter of the exit port is 16 mm. As a light source, a halogen lamp with
filtering of the radiation in the studied spectral range has been used in the
measurements. The diameter of incident light beam on the tissue sample is 3 mm. Scan rate is 2 nm/s.The geometry of the measurements in: (a)
transmittance mode, (b) reflectance mode: (1) the incident beam (diameter 3 mm) (2) the bone sample (3)
the entrance port (square20×20 mm) (4) the transmitted (or diffuse
reflected) radiation (5) the integrating sphere (inner diameter is 100 mm) and (6) the exit
port (diameter 16 mm).
(a)(b)The measurements were carried out at room
temperature about 20°C.Images of cranial porcine bone during optical
clearing under action of pure propylene glycol during 24 hours were obtained by
a digital camera with   crossed polarizers.


### 3.3. Processing of Experimental Data

For processing the experimental data
and determination the change of the optical properties of tissue, the inverse
adding-doubling (IAD) method developed by Prahl et al. [37] has been used. The method is widely used in tissue
optics for processing the experimental data of spectrophotometry with
integrating spheres [38, 39]. An important advantage of the IAD method when
applied to tissue optics is a possibility of rapidly obtaining iterative
solutions with the aid of up-to-date microcomputers [5].This method allows one to determine
the absorption(μa) and the reduced scattering
coefficients (μs′=μs(1−g)) of a tissue from the measured values of the total transmittance
and the diffuse reflectance. Here, μs is the scattering coefficient,
and g is the anisotropy factor of
scattering. In these calculations, the anisotropy factor has been fixed as 0.9,
since this value is typical for the tissue in the visible and NIR spectral
ranges [5].


## 3.1. Materials

For this study, five porcine and ten human
cranial bone samples were used. All bone samples were cortical (or compact) bones. The samples of human cranial bone were obtained frompostmortem examinations. All samples were kept in saline at temperature about 5°C until spectroscopic measurements. The bone
samples were measured during 4–6 hours after
autopsy. The area of the samples was about 25×25 mm2. The thickness of each bone
sample was measured with a micrometer in several points over the sample surface
and averaged. Precision of the single measurement was ±50 μm. Thickness of the samples varied from 1.6±0.1 to 5.0±0.5 mm.In this study,
the commercially available propylene glycol (“Reactive,” Russia) and glycerol (“Ph. Eur.,” Germany)
have been used as clearing agents. The refractive index of propylene glycol and
glycerol has been measured by Abbe refractometer at wavelength 589 nm as 1.43
and 1.47, respectively.


## 3.2. Experimental Setups

The measurements of bone reflectance have been
performed in the spectral range 450–1000 nm using a
commercially available optical multichannel spectrometer LESA-5
(“BioSpec,” Moscow,
Russia) with a fiber-optical probe.  The scheme of the
experimental setup is shown in Figure1. The fiber optical probe consists of
seven optical fibers. All fibers had 200 μm core diameter and a numerical aperture of
0.22. The central fiber delivers incident light to the tissue surface, and six
fibers, placed around the central fiber, collect reflected light. Distance
between the delivering and receiving fibers is 290 μm. As a reference, a white slab BaSO4 with a smooth surface has been used. The sample of bone was placed into cuvette
with the immersion liquid. For spectrophotometric measurements, each sample was
removed from the cuvette. Then the sample was again placed in the cuvette.Figure 1
The geometry of the measurements in reflectance
mode with the spectrometer LESA-5:
(1) halogen lamp (2) fiber-optical probe (3) cuvette with a bone sample (4) grating and photodiode array.The total transmittance and diffuse reflectance
measurements have been performed in the 800–2000 nm
wavelength range using the commercially available spectrophotometer CARY-2415
(“Varian,” Australia)
with an integrating sphere (Figure2). Inner diameter of the sphere is 100 mm, size of the entrance
port is 20×20 mm, and diameter of the exit port is 16 mm. As a light source, a halogen lamp with
filtering of the radiation in the studied spectral range has been used in the
measurements. The diameter of incident light beam on the tissue sample is 3 mm. Scan rate is 2 nm/s.The geometry of the measurements in: (a)
transmittance mode, (b) reflectance mode: (1) the incident beam (diameter 3 mm) (2) the bone sample (3)
the entrance port (square20×20 mm) (4) the transmitted (or diffuse
reflected) radiation (5) the integrating sphere (inner diameter is 100 mm) and (6) the exit
port (diameter 16 mm).
(a)(b)The measurements were carried out at room
temperature about 20°C.Images of cranial porcine bone during optical
clearing under action of pure propylene glycol during 24 hours were obtained by
a digital camera with   crossed polarizers.


## 3.3. Processing of Experimental Data

For processing the experimental data
and determination the change of the optical properties of tissue, the inverse
adding-doubling (IAD) method developed by Prahl et al. [37] has been used. The method is widely used in tissue
optics for processing the experimental data of spectrophotometry with
integrating spheres [38, 39]. An important advantage of the IAD method when
applied to tissue optics is a possibility of rapidly obtaining iterative
solutions with the aid of up-to-date microcomputers [5].This method allows one to determine
the absorption(μa) and the reduced scattering
coefficients (μs′=μs(1−g)) of a tissue from the measured values of the total transmittance
and the diffuse reflectance. Here, μs is the scattering coefficient,
and g is the anisotropy factor of
scattering. In these calculations, the anisotropy factor has been fixed as 0.9,
since this value is typical for the tissue in the visible and NIR spectral
ranges [5].


## 4. Results and Discussion

Result of cranial bone clearing can be
illustrated by Figure3. The complete clearing of the bone took place in about
24 hours. Figure 3 shows images of cranial porcine bone before (a) and after
(b) optical clearing by propylene glycol during 24 hours. The cross drawn on a paper was covered by the bone to
observe the increase of the sample translucence. The thickness of the sample
was 1.5±0.2 mm. It is well seen that before immersion agent action the
tissue was turbid, and the image of cross was not seen. After 24 hours, the
bone became more transparent and the cross image was observed. Besides, blood
vessels being under the bone surface were also clearly seen.The bone sample before (a) and after
(b) optical clearing by propylene glycol during 24 hours. The cross-observed through the bone is marked by an arrow.
(a)(b)In
Figure4, spectra of reflectance of cranial porcine bone during the action of
propylene glycol are presented. In the spectra, three spectral bands
corresponding to blood absorption in the visible range are well seen. They are
the α-band with maximum at 537 nm and the β-band with maximum at 568 nm of oxyhemoglobin
absorption [40]. Decrease of reflectance in the short-wavelength range from 450
to 470 nm can be connected with absorption in long-wavelength wing of the Soret
band of oxyhemoglobin with maximum at 415 nm [40].  The action of immersion
agent produces the decrease of the reflectance of the bone tissue (due to
decreasing light scattering in the tissue) in whole studied spectral range that
indicates that bone becomes more transparent.Figure 4
The
reflectance spectra of the bone sample measured concurrently with
administration of propylene glycol at different time intervals.In the visible spectral range, significant decrease of reflectance of
the sample wetted in propylene glycol in comparison with untreated sample is
observed during the first few minutes. It can be explained by effective
immersion of the upper layers of the sample. Further decrease of the bone
reflectance is caused by penetration of immersion agent into the sample on the
depth of probing (about 100 μm for the given centre-to-centre separation of
the source and detector fibers) and partial matching of refractive indices of
both inorganic matrix and interstitial substance.Figures5 and
6 demonstrate dynamics of optical properties of human cranial bone after
administration of glycerol in spectral range 800–2000 nm
calculated by IAD method on the basis of measured values of the total
transmittance and the diffuse reflectance. Figure 5 presents typical wavelength
dependence of the bone absorption coefficient before glycerol administration
and after an hour of glycerol action. Figure 6 shows typical wavelength
dependence of the reduced scattering coefficient of the bone sample before
glycerol administration and after an hour of glycerol action.Figure 5
The absorption
spectra of bone sample measured before (1)
and after administration of glycerol (2) during an hour. Black circles correspond to data presented
in [41], and open circles correspond to data presented in [42].Figure 6
The reduced
scattering spectra of bone sample measured before (1) and after administration of glycerol (2) during an hour. Black
circles correspond to data presented in [41], and open circles correspond to
data presented in [42].In the near infrared (NIR) spectral range, the absorption bands of water with maximums at 978, 1192, 1464, and 1930 nm are observed [43, 44]. Absorption
band with maximum at 1745 nm corresponds to absorption band of lipids [45]. Comparison
of the data obtained in this study and presented by other authors [41, 42]
shows accordance between them in the spectral range 800–1000 nm.
Insignificant differences between these data can be explained by differences in
the used measuring techniques and the tissue samples preparation and storage
methods [46].It is well seen that administration of
hyperosmotic solutions into cranial bone tissue allows for effective
controlling its optical properties in NIR range. The transparence of the bone
increases due to matching of the refractive indices of the hydroxyapatite
matrix and the interstitial substance. Scatterers in bone tissues are large
osteons with diameter 200–300 μm and cavities between osteons with diameter 50–90 μm as well as nanostructures that cause complex
character of light scattering.Figure6 shows that the reduced scattering
coefficient decreases with wavelength increasing, which, in general,
corresponds to spectral behaviour of the scattering characteristics of most of
tissues [5, 37, 39]. A comparison of the data obtained by us in the spectral
range 800–950 nm with the
data presented by Ugryumova et al. [41] and Firbank et al. [42] shows an
accordance between them (see Figures 5 and 6). Discrepancy between these data
does not prevail 20% [46]. In the spectral range from 1000 to 2000 nm, the
reduced scattering coefficient decreases nonmonotonically with increasing of
wavelength with peaks corresponding to the absorption bands in contrast to
spectral behaviour of bone scattering in the spectral range from 800 to 1000 nm, where the reduced scattering coefficient decreases smoothly with wavelength
increasing. It can be explained by significant decrease of the anisotropy
factor in the range of water absorption bands that produces the increase of the
reduced scattering coefficient and appearance of bands in its spectrum [47, 48].The decrease of both absorption and scattering
coefficients of bone tissue under action of immersion agent (see Figures5, 6)
is not uniform in the studied range. It is well seen that in the wavelength
range from 800 to 1400 nm, absorption coefficient of the bone sample does not
change. In the range from 1400 to 2000, the decrease of the absorption
coefficient is observed in the following way: the average decreasing was about
8% in the range 1400–1800 nm and about
20% in the range 1800–2000 nm. It can
be explained by replacing of bone water by the surrounding glycerol solution.
Since bone absorption properties in the spectral ranges are determined by water
absorption, then replacing of bone water by dehydrogenated glycerol produced
decrease of bone absorption coefficient. Another explanation of the decreasing
of the bone absorption coefficient can be connected with the change of regime
of photon scattering from the multiple to a low-scattering mode. In this case, the
increase of photons free path length should be observed and, thus, more photons
pass the tissue layer almost without absorption.The decrease of tissue reduced scattering
coefficient is observed in whole studied wavelength range. With increasing of
wavelength, the effect also becomes more significant. Analysis of the
experimental results has shown that the average decreasing of the coefficient
was about 2% in the range 800–1100 nm, about 8%
in the range 1100–1400 nm, and
about 30% in the range 1400–2000 nm.Numerous publications discuss the mechanisms of
clearing of soft tissues (i.e., skin,dura
mater, sclera, and others) [5, 15–20, 49, 50]. The
main mechanisms of light scattering reduction induced by immersion agents are
(1) dehydration of tissue constituents, (2) partial replacement of the
interstitial fluid by the immersion substance, and (3) structural modification or
dissociation of collagen [5, 15–20, 49, 50]. We
may hypothesise that dissociation of collagen of bone is not of great
importance for the clearing as it may be in soft tissues because of a rigid
structure of bone tissue. We believe that the main role in the clearing process
has the replacement of water in the interstitial space by the immersion
substance that leads to matching of the refractive indices between tissue
scatterers and ground matter. Both glycerol and propylene glycol, as
hypersosmotic agents, stimulate diffusion flow of bulk water from tissue to
surrounding agent solution which also leads to additional refractive index
matching between scatterers and interstitial space materials. On the other
hand, they prevent total dehydration of tissue due to holding water inside, as
water dilutes initial agent solution [49].The
depth penetration of light into a biological tissue is an important parameter
for the correct determination of the irradiation dose in photothermal and
photodynamic therapy of various diseases [5]. Estimation of the light
penetration depth δ can
be performed with the relation [51]
(1)δ=13μa(μa+μs′).Calculation of
the optical penetration depth has been performed with the absorption and the
reduced scattering coefficient values presented in Figures5 and 6,
respectively, and the result is presented in Figure 7. In Figure 7, it is seen
that in dependence on the wavelength, the penetration depth varies
considerably. The penetration depth is maximal in the spectral ranges 800–900 nm and 1000–1100 nm, where
the optical radiation penetrates up to depths of 0.32–0.37 cm (light
attenuation of 2.7-fold) in intact cranial bone. During
an hour after administration of glycerol, the optical penetration depth
increases by 5% in this spectral range. In the
spectral range 1400–2000 nm, the
penetration depth increases more than 20%.Figure 7
The optical penetration depthδ of light into human bone tissue over the wavelength range from 800
to 2000 nm before (1) and after administration of glycerol (2) during an
hour.


## 5. Conclusion

The changes of optical properties of
the human cranial bone in vitro under action of glycerol have been determined
over the wavelength range 800–2000 nm using the
integrating sphere technique and the inverse adding-doubling method. The
reflectance change of the porcine cranial bone in vitro under action of propylene glycol has been determined
over the wavelength range 450–1000 nm using the
optical multichannel spectrometer.
The decrease of both absorption and reduced scattering coefficient of the
samples up to 20% and 30%, respectively, under action of glycerol has been
demonstrated. The decrease of bone reflectance under action of propylene glycol
was up to 70%.The experiments have shown that administration
of the immersion liquids allows for effective control of bone optical
characteristics that makes bone more transparent, thereby increasing the
ability of light penetration through the tissue. The presented results can be
used in the developing of functional imaging techniques, including OCT and
reflectance spectroscopy.



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*Source: 267867-2008-05-19.xml*