Biopharmaceuticals by Kevin Buckley
The production of active pharmaceutical ingredients (APIs) is currently undergoing its biggest tr... more The production of active pharmaceutical ingredients (APIs) is currently undergoing its biggest transformation in a century. The changes are based on the rapid and dramatic introduction of protein-and macromolecule-based drugs (collectively known as biopharmaceuticals) and can be traced back to the huge investment in biomedical science (in particular in genomics and proteomics) that has been ongoing since the 1970s. Biopharmaceuticals (or biologics) are manufactured using biological-expression systems (such as mammalian, bacterial, insect cells, etc.) and have spawned a large (>E35 billion sales annually in Europe) and growing biopharmaceutical industry (BioPharma). The structural and chemical complexity of biologics, combined with the intricacy of cell-based manufacturing, imposes a huge analytical burden to correctly characterize and quantify both processes (upstream) and products (downstream). In small molecule manufacturing, advances in analytical and computational methods have been extensively exploited to generate process analytical technologies (PAT) that are now used for routine process control, leading to more efficient processes and safer medicines. In the analytical domain, biologic manufacturing is considerably behind and there is both a huge scope and need to produce relevant PAT tools with which to better control processes, and better characterize product macromolecules. Raman spectroscopy, a vibrational spectroscopy with a number of useful properties (nondestructive, non-contact, robust-ness) has significant potential advantages in BioPharma. Key among them are intrinsically high molecular specificity, the ability to measure in water, the requirement for minimal (or no) sample pre-treatment, the flexibility of sampling configurations , and suitability for automation. Here, we review and discuss a representative selection of the more important Raman applications in BioPharma (with particular emphasis on mammalian cell culture). The review shows that the properties of Raman have been successfully exploited to deliver unique and useful analytical solutions, particularly for online process monitoring. However, it also shows that its inherent susceptibility to fluorescence interference and the weakness of the Raman effect mean that it can never be a panacea. In particular, Raman-based methods are intrinsically limited by the chemical complexity and wide analyte-concentration-profiles of cell culture media/bioprocessing broths which limit their use for quantitative analysis. Nevertheless, with appropriate foreknowledge of these limitations and good experimental design, robust analytical methods can be produced. In addition, new technological developments such as time-resolved detectors, advanced lasers, and plasmonics offer potential of new Raman-based methods to resolve existing limitations and/or provide new analytical insights.
Blood by Kevin Buckley
Blood is a bodily fluid that is vital for a number of life functions in animals. To a first appro... more Blood is a bodily fluid that is vital for a number of life functions in animals. To a first approximation, blood is a mildly alkaline aqueous fluid (plasma) in which a large number of free-floating red cells (erythrocytes), white cells (leucocytes), and platelets are suspended. The primary function of blood is to transport oxygen from the lungs to all the cells of the body and move carbon dioxide in the return direction after it is produced by the cells' metabolism. Blood also carries nutrients to the cells and brings waste products to the liver and kidneys. Measured levels of oxygen, nutrients, waste, and electrolytes in blood are often used for clinical assessment of human health. Raman spectroscopy is a non-destructive analytical technique that uses the inelastic scattering of light to provide information on chemical composition , and hence has a potential role in this clinical assessment process. Raman spectroscopic probing of blood components and of whole blood has been ongoing for more than four decades and has proven useful in applications ranging from the understanding of hemoglobin oxygenation, to the discrimination of cancerous cells from healthy lymphocytes, and the forensic investigation of crime scenes. In this paper, we review the literature in the field, collate the published Raman spectroscopy studies of erythrocytes, leucocytes, platelets, plasma, and whole blood, and attempt to draw general conclusions on the state of the field.
Modern transfusion medicine relies on the safe, secure, and cost-effective delivery of donated re... more Modern transfusion medicine relies on the safe, secure, and cost-effective delivery of donated red blood cells (RBCs). Once isolated, RBCs are suspended in a defined additive solution and stored in plastic blood bags in which, over time, they undergo chemical, physiological, and morphological changes that may have a deleterious impact on some patients. Regulations limit the storage period to 42 days and the cells do not routinely undergo analytical testing before use. In this study, we use Raman spectroscopy to interrogate stored RBCs and we identify metabolic and cell-breakdown products, such as haemoglobin and membrane fragments, that build-up in the blood bags as the cells age. Our work points the way to the development of an instrument which could quickly and easily assess the biochemical nature of stored RBC units before they are transfused. Clinical and Biomedical Spectroscopy and Imaging IV, edited by J. Quincy Brown, Volker Deckert, Proc. of SPIE-OSA Vol. 9537, 95370X · © 2015 SPIE-OSA CCC code: 1605-7422/15/$18 · doi: 10.1117/12.2183598 Proc. of SPIE-OSA Vol. 9537 95370X-1 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/17/2015 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx Proc. of SPIE-OSA Vol. 9537 95370X-5 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/17/2015 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx Proc. of SPIE-OSA Vol. 9537 95370X-6 Downloaded From: http://proceedings.spiedigitallibrary.org/ on 08/17/2015 Terms of Use: http://spiedigitallibrary.org/ss/TermsOfUse.aspx
After being separated from (donated) whole blood, red blood cells are suspended in specially form... more After being separated from (donated) whole blood, red blood cells are suspended in specially formulated additive solutions and stored (at 4 °C) in polyvinyl chloride (PVC) blood-bags until they are needed for transfusion. With time, the prepared red cell concentrate (RCC) is known to undergo biochemical changes that lower effectiveness of the transfusion, and thus regulations are in place that limit the storage period to 42 days. At present, RCC is not subjected to analytical testing prior to transfusion. In this study, we use Spatially Offset Raman Spectroscopy (SORS) to probe, non-invasively, the biochemistry of RCC inside sealed blood-bags. The retrieved spectra compare well with conventional Raman spectra (of sampled aliquots) and are dominated by features associated with hemoglobin. In addition to the analytical demonstration that SORS can be used to retrieve RCC spectra from standard clinical blood-bags without breaking the sterility of the system, the data reveal interesting detail about the oxygenation-state of the stored cells themselves, namely that some blood-bags unexpectedly contain measurable amounts of deoxygenated hemoglobin after weeks of storage. The demonstration that chemical information can be obtained non-invasively using spectroscopy will enable new studies of RCC degeneration, and points the way to a Raman-based instrument for quality-control in a blood-bank or hospital setting.
Bone by Kevin Buckley
Spatially offset Raman spectroscopy (SORS) is currently being developed as an in vivo tool for bo... more Spatially offset Raman spectroscopy (SORS) is currently being developed as an in vivo tool for bone disease detection, but to date,
information about the interrogated volume as influenced by the light propagation and scattering characteristics of the bone matrix
is still limited. This paper seeks to develop our general understanding of the sampling depths of SORS in bone specimens as a
function of the applied spatial offset. Equine metacarpal bone was selected as a suitable specimen of compact cortical bone large
enough to allow several thin slices (600 μm) to be cut fromthe dorsal surface. Photon migration at 830-nmexcitation was studied
with five bone slices and a 380-μm-thin polytetrafluoroethylene (PTFE) slice placed consecutively between the layers. To optimize
Raman signal recovery of the PTFE with increasing depthwithin the bone stack required a corresponding increase in spatial offset.
For example, to sample effectively at 2.2-mm depth within the bone required an optimal SORS offset of 7mm. However, with a
7-mm offset, the maximum accessible penetration depth from which the PTFE signal could be still recovered was 3.7mm. These
results provide essential basic information for developing SORS technology for medical diagnostics in general and optimizing
sampling through bone tissue, permitting a better understanding of the relationship between the offset and depth of bone
assessed, in particular. Potential applications include the detection of chemically specific markers for changes in bone matrix
chemistry localized within the tissue and not present in healthy bone.
The tendons in the turkey leg have specific well-defined areas which become mineralized as the an... more The tendons in the turkey leg have specific well-defined areas which become mineralized as the animal ages and they are a thoroughly characterized model system for studying the mineralization process of bone. In this study, nondestructive Raman spectroscopic analysis was used to explore the hypothesis that regions of the turkey tendon that are associated with mineralization exhibit distinct and observable chemical modifications of the collagen prior to the onset of mineralization. The Raman spectroscopy features associated with mineralization were identified by probing (on the micrometer scale) the transition zone between mineralized and nonmineralized regions of turkey leg tendons. These features were then measured in whole tendons and identified in regions of tendon which are destined to become rapidly mineralized around 14 weeks of age. The data show there is a site-specific difference in collagen prior to the deposition of mineral, specifically the amide III band at 1270 cm −1 increases as the collagen becomes more ordered (increased amide III:amide I ratio) in regions that become mineralized compared to collagen destined to remain nonmineralized. If this mechanism were present in materials of different mineral fraction (and thus material properties), it could provide a target for controlling mineralization in metabolic bone disease. A number of bird species have tendons that ossify in specific regions to maximize the stiffness of the organ and thus improve its energy efficiency. 1,2 An example is the extensor tendon of the turkey leg which begins to mineralize proximal and distal to the tarsometatarsal joints when the bird is 10−14 weeks old. 3,4 This mineralization pattern creates two zones of " transition " (1−2 mm wide) between mineralized and nonmineralized tendon; one is one-third of the length of the organ from the proximal end, and the second is one-third of the length of the organ from the distal end (Figure 1). The turkey leg tendon (TLT) is a well-characterized system and has been used to investigate the initiation and progression of mineralization. Electron microscopy of turkey tendons has been used to show initialization of mineralization in the gap regions between the ends of the collagen molecules and X-ray scattering has elucidated the nature of the lateral packing of the collagen molecules. The collagen fibrils become straighter and more tightly packed, with mineral deposited from the center of the tendon toward the proximal end. 5 Biochemical analyses of the collagen from turkey tendons have shown that different post-translational modifications occur in regions of the TLT associated with future mineralization status, that collagen cross-linking in the mineralizing and nonmineralizing regions differ, and that the differences are present in young animals prior to mineralization (the transition zones were not analyzed in that study). 6 Lysyl hydroxylation
Raman Spectroscopy has become an important technique for assessing the composition of excised sec... more Raman Spectroscopy has become an important technique for assessing the composition of excised sections of bone, and
is currently being developed as an in vivo tool for transcutaneous detection of bone disease using spatially offset Raman
spectroscopy (SORS). The sampling volume of the Raman technique (and thus the amount of bone material interrogated
by SORS) depends on the nature of the photon scattering in the probed tissue. Bone is a complex hierarchical material
and to date little is known regarding its diffuse scattering properties which are important for the development and
optimization of SORS as a diagnostic tool for characterizing bone disease in vivo. SORS measurements at 830 nm
excitation wavelength are carried out on stratified samples to determine the depth from which the Raman signal
originates within bone tissue. The measurements are made using a 0.38 mm thin Teflon slice, to give a pronounced and
defined spectral signature, inserted in between layers of stacked 0.60 mm thin equine bone slices. Comparing the stack of
bone slices with and without underlying bone section below the Teflon slice illustrated that thin sections of bone can lose
appreciable number of photons through the unilluminated back surface. The results show that larger SORS offsets lead to
progressively larger penetration depth into the sample; different Raman spectral signatures could be retrieved through up
to 3.9 mm of overlying bone material with a 7 mm offset. These findings have direct impact on potential diagnostic
medical applications; for instance on the detection of bone tumors or areas of infected bone.
Fragility fractures, those fractures which result from low level trauma, have a large and growing... more Fragility fractures, those fractures which result from low level trauma, have a large and growing socio-economic cost in countries
with aging populations. Bone-density-based assessment techniques are vital for identifying populations that are at higher risk of
fracture, but do not have high sensitivity when it comes to identifying individuals who will go on to have their first fragility
fracture. We are developing Spatially Offset Raman Spectroscopy (SORS) as a tool for retrieving chemical information from bone
non-invasively in vivo. Unlike X-ray-based techniques SORS can retrieve chemical information from both the mineral and protein
phases of the bone. This may enable better discrimination between those who will or will not go on to have a fragility fracture
because both phases contribute to bone’smechanical properties. In this study we analyse excised bone with Raman spectroscopy
andmultivariate analysis, and then attempt to look for similar Raman signals in vivo using SORS. We show in the excisedwork that
on average, bone fragments from the necks of fractured femora are more mineralised (by 5–10%) than (cadaveric) non-fractured
controls, but the mineralisation distributions of the two cohorts are largely overlapped. In our in vivo measurements, we observe
similar, but as yet statistically underpowered, differences. After the SORS data (the first SORS measurements reported of healthy
and diseased human cohorts), we identifymethodological developments which will be used to improve the statistical significance
of future experiments and may eventually lead to more sensitive prediction of fragility fractures.
Objective. Osteoarthritis (OA) is a common debilitating disease that results in degeneration of c... more Objective. Osteoarthritis (OA) is a common debilitating disease that results in degeneration of cartilage and bone in the synovial joints. Subtle changes in the molecular structure of the subchondral bone matrix occur and may be associated with cartilage changes. The aim of this study was to explore whether the abnormal molecular changes observed in the matrix of OA subchondral bone can be identified with Raman spectroscopy.
Journal of Archaeological Science, 2015
The Mary Rose was King Henry VIII's flagship before it sank in battle on the 19th July 1545. Over... more The Mary Rose was King Henry VIII's flagship before it sank in battle on the 19th July 1545. Over four hundred men went down with the ship and the environment of the Solent meant their remains were quickly covered in silt. Between 1979 and 1982 the remains of 179 individuals were recovered and examined as part of the excavation of the Mary Rose. The anaerobic environment created by the silt preserved the sailors' bones in remarkable condition and to date much has been learnt about life on the ship. In this study we used Raman spectroscopy (a non-destructive technique), to investigate the chemistry of the human bones, specifically for the identification of disease in archaeological specimens, for the first time. Raman data were collected from five anatomically normal tibiae and five tibiae that were bowed (individuals suspected to have suffered from bone disease in childhood). The data were processed using multivariate analysis (principal component analysis) and results showed the presence of chemical abnormalities in the bowed bones which resulted in the separation of the bones into two clearly defined groups, normal and bowed.
X-ray-based diagnostic techniques, which are by far the most widely used for diagnosing bone diso... more X-ray-based diagnostic techniques, which are by far the most widely used for diagnosing bone disorders and diseases, are largely blind to the protein component of bone. Bone proteins are important because they determine certain mechanical properties of bone and changes in the proteins have been associated with a number of bone diseases. Spatially Offset Raman Spectroscopy (SORS) is a chemically specific analytical technique that can be used to retrieve information noninvasively from both the mineral and protein phases of the bone material in vivo. Here we demonstrate that SORS can be used to detect a known compositional abnormality in the bones of a patient suffering from the genetic bone disorder, osteogenesis imperfecta, a condition which affects collagen. The confirmation of the principle that bone diseases in living patients can be detected noninvasively using SORS points the way to larger studies that focus on osteoporosis and other chronic debilitating bone diseases with large socioeconomic burdens.
In long bones, the functional adaptation of shape and structure occurs along the whole length of ... more In long bones, the functional adaptation of shape and structure occurs along the whole length of the
organ. This study explores the hypothesis that adaptation of bone composition is also site-specific and that the
mineral-to-collagen ratio of bone (and, thus, its mechanical properties) varies along the organ’s length. Raman
spectroscopy was used to map the chemical composition of long bones along their entire length in fine spatial
resolution (1 mm), and then biochemical analysis was used to measure the mineral, collagen, water, and sulfated
glycosaminoglycan content where site-specific differences were seen. The results show that the mineral-to-collagen
ratio of the bone material in human tibiae varies by <5% along the mid-shaft but decreases by >10%
toward the flared extremities of the bone. Comparisons with long bones from other large animals (horses,
sheep, and deer) gave similar results with bone material composition changing across tens of centimeters.
The composition of the bone apatite also varied with the phosphate-to-carbonate ratio decreasing toward
the ends of the tibia. The data highlight the complexity of adaptive changes and raise interesting questions
about the biochemical control mechanisms involved. In addition to their biological interest, the data provide
timely information to researchers developing Raman spectroscopy as a noninvasive tool for measuring
bone composition in vivo (particularly with regard to sampling and measurement protocol).
Arthritis & Rheumatology, 2014
Objective. Osteoarthritis (OA) is a common debilitating disease that results in degeneration of c... more Objective. Osteoarthritis (OA) is a common debilitating disease that results in degeneration of cartilage and bone in the synovial joints. Subtle changes in the molecular structure of the subchondral bone matrix occur and may be associated with cartilage changes. The aim of this study was to explore whether the abnormal molecular changes observed in the matrix of OA subchondral bone can be identified with Raman spectroscopy.
Applied spectroscopy, 2014
Raman spectroscopy was used to show that across 10 cm of diaphyseal (mid-shaft) cortical bone the... more Raman spectroscopy was used to show that across 10 cm of diaphyseal (mid-shaft) cortical bone the phosphate-to-amide I ratio (a measure of the mineral to collagen ratio) can vary by as much as 8%, and the phosphate-to-carbonate ratio (a measure of carbonate inclusion in mineral crystals) by as much as 5%. The data are preliminary but are important because they reveal a spatial variation at a scale that is much larger than many of the spectral maps reported in the literature to date. Thus they illustrate natural variation in chemical composition that could have been overlooked in such studies or could have appeared as an undue error where the overall composition of the bone was investigated. Quantifying the variation in mid-shaft cortical bone at the millimeter/centimeter scale reduces the possibility of natural heterogeneity obscuring the average bone composition, or being mistaken for experimental signal, and results in an improvement in the sampling accuracy analogous to that obta...
Journal of Raman Spectroscopy, 2012
Bone is a composite material comprising a collagen fibril scaffold surrounded by crystals of carb... more Bone is a composite material comprising a collagen fibril scaffold surrounded by crystals of carbonated-hydroxyapatite mineral. It is well established that the relative proportions of mineral and collagen in mature bone are not definite and are adapted in order to 'tune' its mechanical properties. It is not known, however, how the mineral to collagen ratio is controlled. This paper uses Raman spectroscopy (which permits the probing of both the mineral and the collagen phases of bone) to explore the hypothesis that the control mechanism is related to the nature of the collagen and that bones with different levels of mineralisation have qualitatively different collagen. Raman spectra of functionally adapted bones with varying levels of mineralisation are presented and features that indicate the differences in the collagen's secondary structure (amide I band profiles) and post-translational modification (hydroxyproline/proline ratios) are highlighted. The study demonstrates that Raman spectroscopy can provide a means to investigate the mechanisms that control the mineral to collagen ratio of bone. Understanding these mechanisms could pave the way towards the therapeutic alteration of the mineral to collagen ratio and, thus, the control of the mechanical properties of bone.
Frontiers in Endocrinology, 2011
ABSTRACT Osteoarthritis is a common, debilitating disease of joints involving degeneration of car... more ABSTRACT Osteoarthritis is a common, debilitating disease of joints involving degeneration of cartilage and bone. Subtle changes in the molecular structure of subchondral bone precede morphological changes in the osteoarthritic joint. {BR}Raman spectroscopy measures inelastic scattered laser light produced when photons interact with chemical materials. Resultant changes in wavelength form spectra representative of the chemical composition of the given sample. Using the new Raman technology of spatially offset Raman spectroscopy (SORS) it is possible to obtain chemical composition of materials several centimetres beneath a surface. Therefore SORS has the potential to be usefully employed in a clinical context to measure bone beneath cartilage. The aim of our study is to explore the hypothesis that abnormal molecular changes in subchondral bone can be detected with SORS. This extends previous biological work that has shown a high proportion of abnormal homotrimeric collagen in subchondral bone from osteoarthritic hip joints.{BR}Raman spectra were acquired ex vivo from 20 human tibial plateaus with established medial compartment osteoarthritis (radiographic and macroscopic diagnosis). Spectra were analysed to determine the spectral peak height ratio of carbonate-to-phosphate (indicating degree of carbonate substitution in the hydroxyapatite crystals) and phosphate-to-collagen (a measure of mineral/organic ratio). Spectral analyses were compared with biochemical analysis (collagen I alpha chain ratios). A peripheral quantitative computed tomography (pQCT) was used to measure the bone mineral density (BMD) across the samples.{BR}Our spectral data will be presented comparing the medial and lateral sides of the tibial plateau. pQCT results revealed that the subchondral bone of the medial side of the samples was both denser and thicker than that of the lateral side. {BR}The immediate goal is to provide an early indication of joint damage, prior to clinical observations, based on correlating molecular-specific modifications in the subchondral bone. Ultimately our efforts will seek to assess SORS for both characterising and detecting osteoarthritis during its early subclinical phase.
SORS by Kevin Buckley
Journal of Raman Spectroscopy, 2014
The decomposition of spatially offset Raman spectra for complex multilayer systems, such as biolo... more The decomposition of spatially offset Raman spectra for complex multilayer systems, such as biological tissues, requires advanced techniques such as multivariate analyses. Often, in such situations, the decomposition methods can reach their limits of accuracy well before the limits imposed by signal-to-noise ratios. Consequently, more effective reconstruction methods could yield more accurate results with the same data set. In this study we process spatially offset Raman spectroscopy (SORS) data with three different multivariate techniques (band-target entropy minimization (BTEM), multivariate curve resolution and parallel factor analysis (PARAFAC)) and compare their performance when analysing a spectrally challenging plastic model system and an even more challenging problem, the analysis of human bone transcutaneously in vivo. For the in vivo measurements, PARAFAC's requirement of multidimensional orthogonal data is addressed by recording SORS spectra both at different spatial offsets and at different anatomical points, the latter providing added dimensionality through the variation of skin/soft tissue thickness.
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Biopharmaceuticals by Kevin Buckley
Blood by Kevin Buckley
Bone by Kevin Buckley
information about the interrogated volume as influenced by the light propagation and scattering characteristics of the bone matrix
is still limited. This paper seeks to develop our general understanding of the sampling depths of SORS in bone specimens as a
function of the applied spatial offset. Equine metacarpal bone was selected as a suitable specimen of compact cortical bone large
enough to allow several thin slices (600 μm) to be cut fromthe dorsal surface. Photon migration at 830-nmexcitation was studied
with five bone slices and a 380-μm-thin polytetrafluoroethylene (PTFE) slice placed consecutively between the layers. To optimize
Raman signal recovery of the PTFE with increasing depthwithin the bone stack required a corresponding increase in spatial offset.
For example, to sample effectively at 2.2-mm depth within the bone required an optimal SORS offset of 7mm. However, with a
7-mm offset, the maximum accessible penetration depth from which the PTFE signal could be still recovered was 3.7mm. These
results provide essential basic information for developing SORS technology for medical diagnostics in general and optimizing
sampling through bone tissue, permitting a better understanding of the relationship between the offset and depth of bone
assessed, in particular. Potential applications include the detection of chemically specific markers for changes in bone matrix
chemistry localized within the tissue and not present in healthy bone.
is currently being developed as an in vivo tool for transcutaneous detection of bone disease using spatially offset Raman
spectroscopy (SORS). The sampling volume of the Raman technique (and thus the amount of bone material interrogated
by SORS) depends on the nature of the photon scattering in the probed tissue. Bone is a complex hierarchical material
and to date little is known regarding its diffuse scattering properties which are important for the development and
optimization of SORS as a diagnostic tool for characterizing bone disease in vivo. SORS measurements at 830 nm
excitation wavelength are carried out on stratified samples to determine the depth from which the Raman signal
originates within bone tissue. The measurements are made using a 0.38 mm thin Teflon slice, to give a pronounced and
defined spectral signature, inserted in between layers of stacked 0.60 mm thin equine bone slices. Comparing the stack of
bone slices with and without underlying bone section below the Teflon slice illustrated that thin sections of bone can lose
appreciable number of photons through the unilluminated back surface. The results show that larger SORS offsets lead to
progressively larger penetration depth into the sample; different Raman spectral signatures could be retrieved through up
to 3.9 mm of overlying bone material with a 7 mm offset. These findings have direct impact on potential diagnostic
medical applications; for instance on the detection of bone tumors or areas of infected bone.
with aging populations. Bone-density-based assessment techniques are vital for identifying populations that are at higher risk of
fracture, but do not have high sensitivity when it comes to identifying individuals who will go on to have their first fragility
fracture. We are developing Spatially Offset Raman Spectroscopy (SORS) as a tool for retrieving chemical information from bone
non-invasively in vivo. Unlike X-ray-based techniques SORS can retrieve chemical information from both the mineral and protein
phases of the bone. This may enable better discrimination between those who will or will not go on to have a fragility fracture
because both phases contribute to bone’smechanical properties. In this study we analyse excised bone with Raman spectroscopy
andmultivariate analysis, and then attempt to look for similar Raman signals in vivo using SORS. We show in the excisedwork that
on average, bone fragments from the necks of fractured femora are more mineralised (by 5–10%) than (cadaveric) non-fractured
controls, but the mineralisation distributions of the two cohorts are largely overlapped. In our in vivo measurements, we observe
similar, but as yet statistically underpowered, differences. After the SORS data (the first SORS measurements reported of healthy
and diseased human cohorts), we identifymethodological developments which will be used to improve the statistical significance
of future experiments and may eventually lead to more sensitive prediction of fragility fractures.
organ. This study explores the hypothesis that adaptation of bone composition is also site-specific and that the
mineral-to-collagen ratio of bone (and, thus, its mechanical properties) varies along the organ’s length. Raman
spectroscopy was used to map the chemical composition of long bones along their entire length in fine spatial
resolution (1 mm), and then biochemical analysis was used to measure the mineral, collagen, water, and sulfated
glycosaminoglycan content where site-specific differences were seen. The results show that the mineral-to-collagen
ratio of the bone material in human tibiae varies by <5% along the mid-shaft but decreases by >10%
toward the flared extremities of the bone. Comparisons with long bones from other large animals (horses,
sheep, and deer) gave similar results with bone material composition changing across tens of centimeters.
The composition of the bone apatite also varied with the phosphate-to-carbonate ratio decreasing toward
the ends of the tibia. The data highlight the complexity of adaptive changes and raise interesting questions
about the biochemical control mechanisms involved. In addition to their biological interest, the data provide
timely information to researchers developing Raman spectroscopy as a noninvasive tool for measuring
bone composition in vivo (particularly with regard to sampling and measurement protocol).
SORS by Kevin Buckley
information about the interrogated volume as influenced by the light propagation and scattering characteristics of the bone matrix
is still limited. This paper seeks to develop our general understanding of the sampling depths of SORS in bone specimens as a
function of the applied spatial offset. Equine metacarpal bone was selected as a suitable specimen of compact cortical bone large
enough to allow several thin slices (600 μm) to be cut fromthe dorsal surface. Photon migration at 830-nmexcitation was studied
with five bone slices and a 380-μm-thin polytetrafluoroethylene (PTFE) slice placed consecutively between the layers. To optimize
Raman signal recovery of the PTFE with increasing depthwithin the bone stack required a corresponding increase in spatial offset.
For example, to sample effectively at 2.2-mm depth within the bone required an optimal SORS offset of 7mm. However, with a
7-mm offset, the maximum accessible penetration depth from which the PTFE signal could be still recovered was 3.7mm. These
results provide essential basic information for developing SORS technology for medical diagnostics in general and optimizing
sampling through bone tissue, permitting a better understanding of the relationship between the offset and depth of bone
assessed, in particular. Potential applications include the detection of chemically specific markers for changes in bone matrix
chemistry localized within the tissue and not present in healthy bone.
is currently being developed as an in vivo tool for transcutaneous detection of bone disease using spatially offset Raman
spectroscopy (SORS). The sampling volume of the Raman technique (and thus the amount of bone material interrogated
by SORS) depends on the nature of the photon scattering in the probed tissue. Bone is a complex hierarchical material
and to date little is known regarding its diffuse scattering properties which are important for the development and
optimization of SORS as a diagnostic tool for characterizing bone disease in vivo. SORS measurements at 830 nm
excitation wavelength are carried out on stratified samples to determine the depth from which the Raman signal
originates within bone tissue. The measurements are made using a 0.38 mm thin Teflon slice, to give a pronounced and
defined spectral signature, inserted in between layers of stacked 0.60 mm thin equine bone slices. Comparing the stack of
bone slices with and without underlying bone section below the Teflon slice illustrated that thin sections of bone can lose
appreciable number of photons through the unilluminated back surface. The results show that larger SORS offsets lead to
progressively larger penetration depth into the sample; different Raman spectral signatures could be retrieved through up
to 3.9 mm of overlying bone material with a 7 mm offset. These findings have direct impact on potential diagnostic
medical applications; for instance on the detection of bone tumors or areas of infected bone.
with aging populations. Bone-density-based assessment techniques are vital for identifying populations that are at higher risk of
fracture, but do not have high sensitivity when it comes to identifying individuals who will go on to have their first fragility
fracture. We are developing Spatially Offset Raman Spectroscopy (SORS) as a tool for retrieving chemical information from bone
non-invasively in vivo. Unlike X-ray-based techniques SORS can retrieve chemical information from both the mineral and protein
phases of the bone. This may enable better discrimination between those who will or will not go on to have a fragility fracture
because both phases contribute to bone’smechanical properties. In this study we analyse excised bone with Raman spectroscopy
andmultivariate analysis, and then attempt to look for similar Raman signals in vivo using SORS. We show in the excisedwork that
on average, bone fragments from the necks of fractured femora are more mineralised (by 5–10%) than (cadaveric) non-fractured
controls, but the mineralisation distributions of the two cohorts are largely overlapped. In our in vivo measurements, we observe
similar, but as yet statistically underpowered, differences. After the SORS data (the first SORS measurements reported of healthy
and diseased human cohorts), we identifymethodological developments which will be used to improve the statistical significance
of future experiments and may eventually lead to more sensitive prediction of fragility fractures.
organ. This study explores the hypothesis that adaptation of bone composition is also site-specific and that the
mineral-to-collagen ratio of bone (and, thus, its mechanical properties) varies along the organ’s length. Raman
spectroscopy was used to map the chemical composition of long bones along their entire length in fine spatial
resolution (1 mm), and then biochemical analysis was used to measure the mineral, collagen, water, and sulfated
glycosaminoglycan content where site-specific differences were seen. The results show that the mineral-to-collagen
ratio of the bone material in human tibiae varies by <5% along the mid-shaft but decreases by >10%
toward the flared extremities of the bone. Comparisons with long bones from other large animals (horses,
sheep, and deer) gave similar results with bone material composition changing across tens of centimeters.
The composition of the bone apatite also varied with the phosphate-to-carbonate ratio decreasing toward
the ends of the tibia. The data highlight the complexity of adaptive changes and raise interesting questions
about the biochemical control mechanisms involved. In addition to their biological interest, the data provide
timely information to researchers developing Raman spectroscopy as a noninvasive tool for measuring
bone composition in vivo (particularly with regard to sampling and measurement protocol).