Direct Measurement of Chocolate Components Using Dispersive Raman Spectroscopy at 1000 nm Excitation.

Chocolate is a popular food around the world. Making chocolate-based confectionaries involve multiple processing steps including cocoa bean fermentation, cocoa bean roasting, grinding, and then a controlled crystallization, where the processing conditions yields the desirable polymorph V to give chocolate its characteristic snap and texture. Raman spectroscopy is well known as a technique that can provide a non-contact, non-destructive analysis of chemical composition and molecular structure. Yet, excitation in the visible and near-infrared (532-785 nm) has not been possible for dark or milk chocolate because of the samples' overwhelming fluorescence. New technologies enabling Raman spectroscopy closer to shortwave infrared wavelengths, closer to 1000 nm, are likely to reduce fluorescence of chocolate and other highly fluorescent samples. Based on the successes of 1064 nm excitation to understand chocolate blooming, we hypothesized that 1000 nm excitation would also reduce fluorescence and enable Raman spectroscopy in dark and milk chocolates. We used dispersive Raman spectroscopy at 1000 nm to measure white, milk, and dark chocolate and cocoa nibs. The use of 1000 nm excitation effectively reduced fluorescence, enabling qualitative and quantitative Raman spectroscopy directly on chocolate samples. These feasibility studies indicate that 1000 nm Raman spectroscopy can be used to measure chocolate in a laboratory or process environment.

The role of Raman spectroscopy in biopharmaceuticals from development to manufacturing.

Biopharmaceuticals have revolutionized the field of medicine in the types of active ingredient molecules and treatable indications. Adoption of Quality by Design and Process Analytical Technology (PAT) frameworks has helped the biopharmaceutical field to realize consistent product quality, process intensification, and real-time control. As part of the PAT strategy, Raman spectroscopy offers many benefits and is used successfully in bioprocessing from single-cell analysis to cGMP process control. Since first introduced in 2011 for industrial bioprocessing applications, Raman has become a first-choice PAT for monitoring and controlling upstream bioprocesses because it facilitates advanced process control and enables consistent process quality. This paper will discuss new frontiers in extending these successes in upstream from scale-down to commercial manufacturing. New reports concerning the use of Raman spectroscopy in the basic science of single cells and downstream process monitoring illustrate industrial recognition of Raman's value throughout a biopharmaceutical product's lifecycle. Finally, we draw upon a nearly 90-year history in biological Raman spectroscopy to provide the basis for laboratory and in-line measurements of protein quality, including higher-order structure and composition modifications, to support formulation development.

Assessing mechanical behavior of ostrich and equine trabecular and cortical bone based on depth sensing indentation measurements.

Guided bone regeneration surgeries are based on grafting a scaffold in the site to be repaired. The main focus of the scaffold is to provide mechanical support to newly formed blood vessels and cells that will colonize the grafted site, achiving bone regenertation. In this regards, the aim of this study was to characterize the anatomy, structular, surface morphologycal, chemical composition, and nanomechanical properties of ostrich and equine trabecular bone. Ostrich and equine specimens were obtained from a local abattoir and bone was obtained by blunt dissection, n = 5. Tissue bone anatomy and trabecular structure were measured using Computerized Axial Tomography (CAT). Atomic Force Microscopy (AFM) and Energy dispersion spectrometry of X-ray (EDS) were used to examine surface morphology and chemical composition of the trabecular ostrich and equine bone. Mechanical behavior was analysted by nanoindentation. Equine specimens were examined as control. CAT results suggest that in terms of anthropometry, ostrich tarsometatarsus bone is more suitable due to its length is 432.56 ± 3.12 mm vs. the highest human bone structures reported, which femur length is 533.66 ± 18.81 mm. Besides, the low radiodensity in the Hounsfield scale exhibits equine trabecular bone more brittle (Av = 1538.4 ± 0.9) than ostrich trabecular bone (Av = 462.1 ± 1.5). EDS showed a slight variation of the element Calcium (Ca2+) ranging from 20% to 25.5% wt in equine bone; the Ca2+ content variation is consistent with the ring-shaped morphology, while in ostrich bone the chemical composition is homogeneous. The elastic modulus, nanohardness (E = 5.3 ± 0.7 GPa, H = 220 ± 10 MPa) and average roughness (Ra = 207 nm) are similar to the human trabecular bone which could reduce the stress shielding, all of these findings suggest that ostrich bone can be promising for native tissue scaffolds for mechanically demanding applications. This research makes innovative contributions to science and provides a framework, which will allow us to address future biomedical tests, and rapidly identify promising organic and sustainable waste for tissue scaffold.

Optical study to identify and quantify capsaicin in optical window.

Capsaicin is an active compound in hot peppers. It has been studied for its health benefits for humans. Optical spectroscopy is an important tool for determining the optical properties or chemical composition of matter. The aim of this research is proposing an optical method to identify and quantify capsaicin in the visible range. To achieve this goal, we combined absorption and diffuse reflectance spectroscopy techniques to compute the extinction coefficient. Moreover, the concentration of the analytes was determined using the optical properties of capsaicin. Our method is a promising tool for developing a classification of capsaicin according to its percentage in chilies. The extinction coefficients are reported for 507nm and 663nm, which are the most significative. In addition, the coefficients to build the mathematical model for capsaicin are reported for Kubelka-Munk model. Finally, a comparison between capsaicin vs chilies spectra was obtained to identify spectral response. Diffuse reflectance signals allowed the identification of capsaicin and opened the possibility of this fast and easy to do method for classification and quantification of bioactive compounds.

Raman spectroscopy as a process analytical technology for pharmaceutical manufacturing and bioprocessing.

Adoption of Quality by Design (QbD) principles, regulatory support of QbD, process analytical technology (PAT), and continuous manufacturing are major factors effecting new approaches to pharmaceutical manufacturing and bioprocessing. In this review, we highlight new technology developments, data analysis models, and applications of Raman spectroscopy, which have expanded the scope of Raman spectroscopy as a process analytical technology. Emerging technologies such as transmission and enhanced reflection Raman, and new approaches to using available technologies, expand the scope of Raman spectroscopy in pharmaceutical manufacturing, and now Raman spectroscopy is successfully integrated into real-time release testing, continuous manufacturing, and statistical process control. Since the last major review of Raman as a pharmaceutical PAT in 2010, many new Raman applications in bioprocessing have emerged. Exciting reports of in situ Raman spectroscopy in bioprocesses complement a growing scientific field of biological and biomedical Raman spectroscopy. Raman spectroscopy has made a positive impact as a process analytical and control tool for pharmaceutical manufacturing and bioprocessing, with demonstrated scientific and financial benefits throughout a product's lifecycle.

Using Raman spectroscopy to characterize biological materials.

Raman spectroscopy can be used to measure the chemical composition of a sample, which can in turn be used to extract biological information. Many materials have characteristic Raman spectra, which means that Raman spectroscopy has proven to be an effective analytical approach in geology, semiconductor, materials and polymer science fields. The application of Raman spectroscopy and microscopy within biology is rapidly increasing because it can provide chemical and compositional information, but it does not typically suffer from interference from water molecules. Analysis does not conventionally require extensive sample preparation; biochemical and structural information can usually be obtained without labeling. In this protocol, we aim to standardize and bring together multiple experimental approaches from key leaders in the field for obtaining Raman spectra using a microspectrometer. As examples of the range of biological samples that can be analyzed, we provide instructions for acquiring Raman spectra, maps and images for fresh plant tissue, formalin-fixed and fresh frozen mammalian tissue, fixed cells and biofluids. We explore a robust approach for sample preparation, instrumentation, acquisition parameters and data processing. By using this approach, we expect that a typical Raman experiment can be performed by a nonspecialist user to generate high-quality data for biological materials analysis.

Parathyroid hormone attenuates radiation-induced increases in collagen crosslink ratio at periosteal surfaces of mouse tibia.

As part of our ongoing efforts to understand underlying mechanisms contributing to radiation-associated bone fragility and to identify possible treatments, we evaluated the longitudinal effects of parathyroid hormone (PTH) treatment on bone quality in a murine model of limited field irradiation. We hypothesized PTH would mitigate radiation-induced changes in the chemical composition and structure of bone, as measured by microscope-based Raman spectroscopy. We further hypothesized that collagen crosslinking would be especially responsive to PTH treatment. Raman spectroscopy was performed on retrieved tibiae (6-7/group/time point) to quantify metrics associated with bone quality, including: mineral-to-matrix ratio, carbonate-to-phosphate ratio, mineral crystallinity, collagen crosslink (trivalent:divalent) ratio, and the mineral and matrix depolarization ratios. Irradiation disrupted the molecular structure and orientation of bone collagen, as evidenced by a higher collagen crosslink ratio and lower matrix depolarization ratio (vs. non-irradiated control bones), persisting until 12weeks post-irradiation. Radiation transiently affected the mineral phase, as evidenced by increased mineral crystallinity and mineral-to-matrix ratio at 4weeks compared to controls. Radiation decreased bone mineral depolarization ratios through 12weeks, indicating increased mineral alignment. PTH treatment partially attenuated radiation-induced increases in collagen crosslink ratio, but did not restore collagen or mineral alignment. These post-radiation matrix changes are consistent with our previous studies of radiation damage to bone, and suggest that the initial radiation damage to bone matrix has extensive effects on the quality of tissue deposited thereafter. In addition to maintaining bone quality, preventing initial radiation damage to the bone matrix (i.e. crosslink ratio, matrix orientation) may be critical to preventing late-onset fragility fractures.

Raman spectroscopy for label-free identification of calciphylaxis.

Calciphylaxis is a painful, debilitating, and premorbid condition, which presents as calcified vasculature and soft tissues. Traditional diagnosis of calciphylaxis lesions requires an invasive biopsy, which is destructive, time consuming, and often leads to exacerbation of the condition and infection. Furthermore, it is difficult to find small calcifications within a large wound bed. To address this need, a noninvasive diagnostic tool may help clinicians identify ectopic calcified mineral and determine the disease margin. We propose Raman spectroscopy as a rapid, point-of-care, noninvasive, and label-free technology to detect calciphylaxis mineral. Debrided calciphylactic tissue was collected from six patients and assessed by microcomputed tomography (micro-CT). Micro-CT confirmed extensive deposits in three specimens, which were subsequently examined with Raman spectroscopy. Raman spectra confirmed that deposits were consistent with carbonated apatite, consistent with the literature. Raman spectroscopy shows potential as a noninvasive technique to detect calciphylaxis in a clinical environment.

Next-generation Raman tomography instrument for non-invasive in vivo bone imaging.

Combining diffuse optical tomography methods with Raman spectroscopy of tissue provides the ability for in vivo measurements of chemical and molecular characteristics, which have the potential for being useful in diagnostic imaging. In this study a system for Raman tomography was developed and tested. A third generation microCT coupled system was developed to combine 10 detection fibers and 5 excitation fibers with laser line filtering and a Cytop reference signal. Phantom measurements of hydroxyapatite concentrations from 50 to 300 mg/ml had a linear response. Fiber placement and experiment design was optimized using cadaver animals with live animal measurements acquired to validate the systems capabilities. Promising results from the initial animal experiments presented here, pave the way for a study of longitudinal measurements during fracture healing and the scaling of the Raman tomography system towards human measurements.

Raman spectroscopy of soft musculoskeletal tissues.

Tendon, ligament, and joint tissues are important in maintaining daily function. They can be affected by disease, age, and injury. Slow tissue turnover, hierarchical structure and function, and nonlinear mechanical properties present challenges to diagnosing and treating soft musculoskeletal tissues. Understanding these tissues in health, disease, and injury is important to improving pharmacologic and surgical repair outcomes. Raman spectroscopy is an important tool in the examination of soft musculoskeletal tissues. This article highlights exciting basic science and clinical/translational Raman studies of cartilage, tendon, and ligament.

Characterization of biofluids prepared by sessile drop formation.

Sessile drop formation, also called drop deposition, has been studied as a potential medical diagnostic, but the effects of complex biofluid rheology on the final deposition pattern are not well understood. We studied two model biofluids, blood plasma and synovial fluid, when deposited onto slightly hydrophilic substrates forming a contact angle of 50-90°. Drops were imaged during the evaporation process and geometric properties of the drop, such as contact angle and drop height, were calculated from the images. The resulting dried biofluid drops were then examined using light microscopy and Raman spectroscopy to assess morphological and chemical composition of the dried drop. The effect of substrate contact angle (surface wetting) and fluid concentration was examined. We found that when biofluids are deposited onto slightly hydrophilic surfaces, with a contact angle of 50-90°, a ring-shaped deposit was formed. Analysis of the drying drop's geometric properties indicates that biofluid dynamics follow the piling model of drop formation, as proposed by Deegan et al. The final deposition pattern varied with substrate surface and concentration, as shown by light microscopy photos of dried drops. The chemical composition of the outer ring was minimally affected by substrate surface, but the spatial heterogeneity of protein distribution within the ring varied with concentration. These results indicate that biofluid drop deposition produces ring-shaped deposits which can be examined by multiple analytical techniques.

Alterations to bone mineral composition as an early indication of osteomyelitis in the diabetic foot.

OBJECTIVE: Osteomyelitis in the diabetic foot is a major risk factor for amputation, but there is a limited understanding of early-stage infection, impeding limb-preserving diagnoses. We hypothesized that bone composition measurements provide insight into the early pathophysiology of diabetic osteomyelitis. RESEARCH DESIGN AND METHODS: Compositional analysis by Raman spectroscopy was performed on bone specimens from patients with a clinical diagnosis of osteomyelitis in the foot requiring surgical intervention as either a biopsy (n = 6) or an amputation (n = 11). RESULTS: An unexpected result was the discovery of pathological calcium phosphate minerals in addition to normal bone mineral. Dicalcium phosphate dihydrate, also called brushite, and uncarbonated apatite were found to be exclusively associated with infected bone. CONCLUSIONS: Compositional measurements provided a unique insight into the pathophysiology of osteomyelitis in diabetic foot ulcers. At-patient identification of pathological minerals by Raman spectroscopy may serve as an early-stage diagnostic approach.

Effects of pathology dyes on Raman bone spectra.

We report an overlooked source of artifacts for clinical specimens, where unexpected and normally negligible contaminants can skew the interpretation of results. During an ongoing study of bone fragments from diabetic osteomyelitis, strong Raman signatures were found, which did not correspond with normal bone mineral or matrix. In a bone biopsy from the calcaneus of a patient affected by diabetic osteomyelitis, Raman microspectroscopic analysis revealed regions with both abnormal mineral and degraded collagen in addition to normal bone. Additional bands indicated a pathological material. Stenotrophomonas maltophilia was identified in the wound culture by independent microbiologic examination. We initially assigned the unusual bands to xanthomonadin, a bacterial pigment from S. maltophilia. However, the same bands were also found more than a year later on a second specimen that had been noticeably contaminated with pathology marking dye. Drop deposition/Raman spectroscopy of commonly used pathology dyes revealed that a blue tissue-marking dye was responsible for the unusual bands in both specimens, even in the first specimen where there was no visible evidence of contamination.

Biomedical tissue phantoms with controlled geometric and optical properties for Raman spectroscopy and tomography.

To support the translation of Raman spectroscopy into clinical applications, synthetic models are needed to accurately test, optimize and validate prototype fiber optic instrumentation. Synthetic models (also called tissue phantoms) are widely used for developing and testing optical instrumentation for diffuse reflectance, fluorescence, and Raman spectroscopies. While existing tissue phantoms accurately model tissue optical scattering and absorption, they do not typically model the anatomic shapes and chemical composition of tissue. Because Raman spectroscopy is sensitive to molecular composition, Raman tissue phantoms should also approximate the bulk tissue composition. We describe the fabrication and characterization of tissue phantoms for Raman tomography and spectroscopy. These phantoms have controlled chemical and optical properties, and also multilayer morphologies which approximate the appropriate anatomic shapes. Tissue phantoms were fabricated to support on-going Raman studies by simulating the human wrist and rat leg. Surface meshes (triangle patch models) were generated from computed tomography (CT) images of a human arm and rat leg. Rapid prototyping was used to print mold templates with complex geometric patterns. Plastic casting techniques used for movie special effects were adapted to fabricate molds from the rapid prototypes, and finally to cast multilayer gelatin tissue phantoms. The gelatin base was enriched with additives to model the approximate chemistry and optical properties of individual tissue layers. Additional studies were performed to determine optimal casting conditions, phantom stability, layer delamination and chemical diffusion between layers. Recovery of diffuse reflectance and Raman spectra in tissue phantoms varied with probe placement. These phantoms enable optimization of probe placement for human or rat studies. These multilayer tissue phantoms with complex geometries are shown to be stable, with minimal layer delamination and chemical diffusion.

Fiber-optic Raman spectroscopy of joint tissues.

In this study, we report adaptation of Raman spectroscopy for arthroscopy of joint tissues using a custom-built fiber-optic probe. Differentiation of healthy and damaged tissue or examination of subsurface tissue, such as subchondral bone, is a challenge in arthroscopy because visual inspection may not provide sufficient contrast. Discrimination of healthy versus damaged tissue may be improved by incorporating point spectroscopy or hyperspectral imaging into arthroscopy where the contrast is based on the molecular structure or chemical composition. Articular joint surfaces of knee cadaveric human tissue and tissue phantoms were examined using a custom-designed Raman fiber-optic probe. Fiber-optic Raman spectra were compared against reference spectra of cartilage, subchondral bone and cancellous bone collected using Raman microspectroscopy. In fiber-optic Raman spectra of the articular surface, there was an effect of cartilage thickness on recovery of signal from subchondral bone. At sites with intact cartilage, the bone mineralization ratio decreased but there was a minimal effect in the bone mineral chemistry ratios. Tissue phantoms were prepared as experimental models of the osteochondral interface. Raman spectra of tissue phantoms suggested that optical scattering of cartilage has a large effect on the relative cartilage and bone signal. Finite element analysis modeling of light fluence in the osteochondral interface confirmed experimental findings in human cadaveric tissue and tissue phantoms. These first studies demonstrate the proof of principle for Raman arthroscopic measurement of joint tissues and provide a basis for future clinical or animal model studies.

Minor distortions with major consequences: correcting distortions in imaging spectrographs.

Projective transformation is a mathematical correction (implemented in software) used in the remote imaging field to produce distortion-free images. We present the application of projective transformation to correct minor alignment and astigmatism distortions that are inherent in dispersive spectrographs. Patterned white-light images and neon emission spectra were used to produce registration points for the transformation. Raman transects collected on microscopy and fiber-optic systems were corrected using established methods and compared with the same transects corrected using the projective transformation. Even minor distortions have a significant effect on reproducibility and apparent fluorescence background complexity. Simulated Raman spectra were used to optimize the projective transformation algorithm. We demonstrate that the projective transformation reduced the apparent fluorescent background complexity and improved reproducibility of measured parameters of Raman spectra. Distortion correction using a projective transformation provides a major advantage in reducing the background fluorescence complexity even in instrumentation where slit-image distortions and camera rotation were minimized using manual or mechanical means. We expect these advantages should be readily applicable to other spectroscopic modalities using dispersive imaging spectrographs.

Raman spectroscopy of synovial fluid as a tool for diagnosing osteoarthritis.

For many years, viscosity has been the primary method used by researchers in rheumatology to assess the physiochemical properties of synovial fluid in both normal and osteoarthritic patients. However, progress has been limited by the lack of methods that provide multiple layers of information, use small sample volumes, and are rapid. Raman spectroscopy was used to assess the biochemical composition of synovial fluid collected from 40 patients with clinical evidence of knee osteoarthritis (OA) at the time of elective surgical treatment. Severity of knee osteoarthritis was assessed by a radiologist using Kellgren/Lawrence (K/L) scores from knee joint x rays, while light microscopy and Raman spectroscopy were used to examine synovial fluid (SF) aspirates (2 to 10 microL), deposited on fused silica slides. We show that Raman bands used to describe protein secondary structure and content can be used to detect changes in synovial fluid from osteoarthritic patients. Several Raman band intensity ratios increased significantly in spectra collected from synovial fluid in patients with radiological evidence of moderate-to-severe osteoarthritis damage. These ratios can be used to provide a "yes/no" damage assessment. These studies provide evidence that Raman spectroscopy would be a suitable candidate in the evaluation of joint damage in knee osteoarthritis patients.

Automated Raman Spectral Preprocessing of Bone and Other Musculoskeletal Tissues.

Raman spectroscopy of bone is complicated by fluorescence background and spectral contributions from other tissues. Full utilization of Raman spectroscopy in bone studies requires rapid and accurate calibration and preprocessing methods. We have taken a step-wise approach to optimize and automate calibrations, preprocessing and background correction. Improvements to manual spike removal, white light correction, software image rotation and slit image curvature correction are described. Our approach is concisely described with a minimum of mathematical detail.

Effect of conformation and drop properties on surface-enhanced Raman spectroscopy of dried biopolymer drops.

Biofluids are complex solutions consisting of small ions and large biopolymers such as DNA, proteins, or proteoglycans. Biopolymers affect fluid properties but their effect on drop deposition has not been examined. Hyaluronic acid (HA), an important component in synovial fluid, was chosen as a model biopolymer, and examined using surface-enhanced Raman spectroscopy (SERS). Nanoliter volumes of HA solutions were dried onto a patterned SERS substrate and spectra were collected from the dried hyaluronic acid drops with a near-infrared Raman microscope. Characteristic hyaluronic acid bands were examined. Capillary viscometry measured properties of HA solutions, and entanglement behavior was also modeled using scaling theory principles. Viscosity measurements were incorporated into models of suspended particle droplets to account for the effect of inter-chain attraction on droplet formation. Microscope images were used to evaluate the shape of the dried drop. Relative drop thickness was estimated from concentric rings found at drop edges using established models of light interference by thin films. We found SERS spectra were sensitive not only to polymer conformation, but also to type of deposition (ring versus uniform), and the thickness of the resulting deposition. These data suggest an approach to elucidate the effects of biopolymers and dehydrated biofluids on SERS analysis.