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.
Raman spectroscopy can non-destructively measure properties of bone related to mineral density, mineral composition,
and collagen composition. Bone properties can be measured through the skin in animal and human subjects, but
correlations between the transcutaneous and exposed bone measurements have only been reported for human cadavers.
In this study, we examine human subjects to collect measurements transcutaneously, on surgically exposed bone, and on
recovered bone fragments. This data will be used to demonstrate in vivo feasibility and to compare transcutaneous and
exposed Raman spectroscopy of bone. A commercially available Raman spectrograph and optical probe operating at
785 nm excitation are used for the in vivo measurements. Requirements for applying Raman spectroscopy during a
surgery are also discussed.
Allografts and other bone-grafts are frequently used for a variety of reconstructive approaches in orthopaedic surgery.
However, successful allograft incorporation remains uncertain. Consequently, there is significant need for methods to
monitor the fate of these constructs. Only few noninvasive methods can fully assess the progress of graft incorporation
and to provide information on the metabolic status of the graft, such as the mineral and matrix composition of the
regenerated-tissue that may provide early indications of graft success or failure. For example, Computed-tomography
and MRI provide information on the morphology of the graft/host interface. Limited information is also available from
DXA. To address this challenge, we present here the implementation of a noninvasive Raman spectroscopy technique for
in-vivo assessment of allograft incorporation in animal-model.
In an animal use committee approved osseointegration experiment, a 3mm defect is created in rat's tibia. The defect is
reconstructed using auto or allograft and Raman spectra are collected at several time-points during healing using an array
of optical-fibers in contact with the skin of the rat over the tibia while the rat is anaesthetized. The array allows excitation
and collection of Raman spectra through the skin at various positions around the tibia. Raman parameters such as
mineral/matrix, carbonate/phosphate and cross-linking are recovered and monitored. The system is calibrated against
locally-constructed phantoms that mimic the morphology, optics and spectroscopy of the rat. This new technology
provides a non-invasive method for in-vivo assessment of bone-graft incorporation in animal-models and can be adapted
for similar study in human subjects.
Recovery of Raman or Fluorescence signatures from within thin tissues benefits from model-based estimation of where
the signal came from, especially if the signal passes through layers in which the absorption or scattering signatures
distort the signal. Estimation of the signal strength requires appropriate normalization or model-based recovery, but the
key to achieving good results is a good model of light transport. While diffusion models are routinely used for optical
tomography of tissue, there's some thought that more precise radiation transport modeling is required for accurate
estimation. However, diffusion is often used for small animal imaging, because it's a practical approach, which doesn't
require knowledge of the scatter phase function at each point in the tissue. The question asked in this study is, whether
experimentally acquired data in small volumes such as a rodent leg can be accurately modeled and reconstructed using
diffusion theory. This study uses leg geometries extracted from animal CT scans and liquid phantoms to study the
diffusion approximations. The preliminary results show that under certain conditions the collected data follows the
expected trend.
Raman spectroscopic measurement of bone composition has shown promise as a medical diagnostic by measuring the
molecular composition of the bone mineral and matrix. We previously demonstrated proof-of-principle transcutaneous
Raman spectroscopy bone measurements in human cadavers. In this paper, we discuss further optimization of the
instrumental configuration for efficient collection of bone signal using contact fiber-optic probe designs. To optimize
collection of Raman signal through overlaying soft tissue, novel geometrically-accurate tissue phantoms were prepared.
MRI and CT images of the human cadaveric specimens were used to create solid tissue phantoms with accurate
geometric dimensions. In these tissue phantoms, optical properties can be varied systematically. Raman spectra of the
prepared tissue phantoms were used to optimize the positions of the fibers in the fiber optic system, and the laser
illumination sequence in the measurements. Three fiber optic probes were developed and tested with both novel tissue
phantoms and human cadaveric specimens. The contact fiber optic probes were developed for arthroscopic
measurements of joints, for transcutaneous measurements of bone in situ, and for contact measurements of exposed
bone. By coupling the fiber optic probe to an imaging spectrograph, spectra were collected simultaneously at many
positions on the tissue. Furthermore, spectra were collected with several different excitation laser patterns to enhance the
effective spatial resolution of the measurements. Finally, a series of improvements were made in the data preprocessing
to improve the recovered spectral signal. Together, these modifications improve signal-to-noise and spatial resolution.
As part of our ongoing assessment of bone tissue composition and structure, we report the first experimental protocols of
a prospective study to investigate the potential of using Raman spectroscopy to diagnose and predict skeletal fragility in
postmenopausal osteoporosis patients. This multi-center study will assess several potential spectroscopic and X-ray
based diagnostic techniques. One hundred and twenty participants will be enrolled in this five year study and the
investigators will be blinded to information concerning patient history and status. Iliac crest bone biopsy specimens are
provided with no identifying information except a patient study number. Our team will use micro-computed tomography
(micro-CT) to identify regions of interest in both cortical and cancellous bone from specimens delivered to us. Raman
mapping will be performed using a line-focused 785 nm laser in order to obtain local and averaged values on several
spectroscopic metrics of bone quality. These metrics include carbonate/phosphate and phosphate/matrix ratios. Results
from an initial set of biopsies will be presented. Protocols for obtaining measurements are discussed, with emphasis on
the challenges presented by the use of fixed and polymer embedded specimens. These protocols are illustrated will data
from a biopsy specimen.
Light can be coupled into imaging spectrographs through the use of fiber-optic bundles. Ideally, the collected
spectra from adjacent optical fibers should be resolved and independent. However, this assumption breaks down if a
partial overlap of adjacent fibers on the detector CCD results from either diffraction or uncorrected monochromatic
aberrations. In addition, spectral mixing can be caused by optical cross-talk among tightly packed fibers, particularly if
the fiber-optic buffer has been removed in order to use the CCD area more efficiently and increase the linear fiber
packing density. These coupling effects can become sources of systematic error, especially when fiber bundles are used
for imaging or when branches of a multi-leg fiber array are interrogating different samples. Coupling errors can mix
spectra from different spatial regions of the sample. In this paper, the mixing in fiber bundles will be assessed, and
mathematical methods to resolve overlapped signals and correct for signal mixing will be discussed. Using data
processing to correct for optical coupling errors, instead of physically changing the spacing between fibers in the bundle,
allows for the efficient use of the limited spectrograph detector area by having a larger number of fibers.
We describe the use of Raman spectroscopy to investigate synovial fluid drops deposited onto fused silica microscope slides. This spectral information can be used to identify chemical changes in synovial fluid associated with osteoarthritis (OA) damage to knee joints. The chemical composition of synovial fluid is predominately proteins (enzymes, cytokines, or collagen fragments), glycosaminoglycans, and a mixture of minor components such as inorganic phosphate crystals. During osteoarthritis, the chemical, viscoelastic and biological properties of synovial fluid are altered. A pilot study was conducted to determine if Raman spectra of synovial fluid correlated with radiological scoring of knee joint damage. After informed consent, synovial fluid was drawn and x-rays were collected from the knee joints of 40 patients. Raman spectra and microscope images were obtained from the dried synovial fluid drops using a Raman microprobe and indicate a coarse separation of synovial fluid components. Individual protein signatures could not be identified; Raman spectra were useful as a general marker of overall protein content and secondary structure. Band intensity ratios used to describe protein and glycosaminoglycan structure were used in synovial fluid spectra. Band intensity ratios of Raman spectra indicate that there is less ordered protein secondary structure in synovial fluid from the damage group. Combination of drop deposition with Raman spectroscopy is a powerful approach to examining synovial fluid for the purposes of assessing osteoarthritis damage.
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.
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