Nicole J. Crane

Band-Target Entropy Minimization (BTEM) Applied to Hyperspectral Raman Image Data:

Band-target entropy minimization (BTEM) has been applied to extraction of component spectra from hyperspectral Raman images. In this method singular value decomposition is used to calculate the eigenvectors of the spectroscopic image data set. Bands in non-noise eigenvectors that would normally be used for recovery of spectra are examined for localized spectral features. For a targeted (identified) band, information entropy minimization or a closely related algorithm is used to recover the spectrum containing this feature from the non-noise eigenvectors, plus the next 5–30 eigenvectors, in which noise predominates. Tests for which eigenvectors to include are described. The method is demonstrated on one synthesized Raman image data set and two bone tissue specimens. By inclusion of small amounts of signal that would be unused in other methods, BTEM enables the extraction of a larger number of component spectra than are otherwise obtainable. An improvement in signal/noise ratio of the recovered spectra is also obtained.

Identifying chemical changes in subchondral bone taken from murine knee joints using Raman spectroscopy.

Application of Raman spectroscopy to analysis of subchondral bone is described. The effect of cartilage health on subchondral bone has been widely studied using radiological and histological methods; however, there is no method to directly assay mineral components. We present Raman spectra of femur condyles and observe mineral bands that arise from the subchondral bone. In two separate experiments, transgenic mouse models of early-onset osteoarthritis (OA) and lipoatrophy were compared to tissue from wild-type mice. Raman spectroscopy was used to identify chemical changes in the mineral of subchondral bone that may accompany or precede morphological changes that can be observed by histology. The transgenic mice were compared to age-matched wild-type mice. Subtle alterations in the mineral or collagen matrix were observed by Raman spectroscopy using established Raman markers such as the carbonate-to-phosphate ratio, mineral-to-matrix ratio (MTMR), and amide I ratio. The Raman microscope configuration enabled rapid collection of Raman spectra from the mineralized layer that lies under an intact layer of non-mineralized articular cartilage. The effect of the cartilage layer on collection of spectra is discussed. The technique proposed is capable of providing insight into the chemical changes that occur in subchondral bone on a molecular level.

Raman imaging demonstrates FGF2-induced craniosynostosis in mouse calvaria.

Craniosynostosis is a severe craniofacial disease where one or more sutures, the fibrous tissue that lies between the cranial bones, fuses prematurely. Some craniosynostosis syndromes are known to be caused by mutations in fibroblast growth factor (FGF) receptors. Mutated FGF receptors are thought to cause constitutive signaling. In this study, heparin acrylic beads released fibroblast growth factor 2 (FGF2) to mimic constitutive signaling by mutated receptors, delivering FGF2 in addition to already existing normal tissue amounts. Fetal day 18.5 mouse sutures were treated with FGF2-soaked beads and cultured in serum free media for 48 h. We have shown previously that this treatment leads to fusion and increased Msx2 expression, but here we use near-infrared Raman imaging to simultaneously examine the mineral components and matrix components of cranial tissue while providing light microscopic spatial information. FGF2-treated mouse sutures show increased v1 phosphate and v1 carbonate bandwidths, indicating a slightly chemically modified mineral being rapidly deposited. In addition, FGF2-treated mouse sutures show a marked increase in mineral-to-matrix ratios compared to control mouse sutures, typical of increased mineralization.

Compatibility of staining protocols for bone tissue with Raman imaging.

We report the use of Raman microscopy to image mouse calvaria stained with hematoxylin, eosin and toluidine blue. Raman imaging of stained specimens allows for direct correlation of histological and spectral information. A line-focus 785 nm laser imaging system with specialized near-infrared (NIR) microscope objectives and CCD detector were used to collect approximately 100 × 450 µm Raman images. Principal components analysis, a multivariate analysis technique, was used to determine whether the histological stains cause spectral interference (band shifts or intensity changes) or result in thermal damage to the examined tissue. Image analysis revealed factors for tissue components and the embedding medium, glycol methacrylate, only. Thus, Raman imaging proved to be compatible with histological stains such as hematoxylin, eosin and toluidine blue.

Spectral imaging of mouse calvaria undergoing craniosynstosis

Craniosynostosis, the premature fusion of the skull bones at the sutures, is the second most common human birth defect that affects the face and skull. The top most flat bones that comprise the skull, or calvaria, are most often affected. We previously showed that treatment of mouse calvaria with FGF2-soaked beads leads to craniosynostosis. In this study we treated mouse calvaria with FGF2-soaked beads and then used Raman imaging to demonstrate the spatial distribution of apatitic mineral and matrix in the sutures. There was no difference between FGF2 treated and control calvaria in the type of mineral produced (a lightly carbonated apatite), however we did observe increased mineral deposition in FGF2 treated calvaria. Raman imaging has great promise to detect the earliest mineral and matrix changes that occur in craniosynostosis.

Study of localization of response to fibroblast growth factor-2 in murine calvaria using Raman spectroscopic imaging

Raman spectral imaging provides the means to study spatially localized response to controlled external perturbations to tissue specimens with light microscopy resolution. We discuss use of heparin-acrylic microbeads soaked with the protein fibroblast growth factor-2 (FGF2). Microbeads containing FGF2 are placed in murine calvarial tissue and stimulate abnormally rapid mineralization. The tissue response simulated the effects of craniosynostosis, a birth defect occurring in 1 in 2400 live births. We describe Raman imaging measurements of the spatial distribution of apatitic mineral and matrix (predominantly type I collagen) from normal murine calvarial tissue and murine calvarial tissue modeling craniosynostosis. We also discuss spectroscopic evaluation of the state of the mineral induced by the FGF2 beads.

Spectral imaging of mouse skulls undergoing craniosynostosis

Craniosynostosis, the premature fusion of the skull bones at the sutures, is the second most common human birth defect that affects the face and skull. The top most flat bones that comprise the skull, or calvaria, are most often affected. We previously showed that treatment of mouse calvaria with FGF2-soaked beads leads to craniosynostosis. In this study we treated mouse calvaria with FGF2-soaked beads and then used Raman imaging to demonstrate the spatial distribution of apatitic mineral and matrix in the sutures. There was no difference between FGF2 treated and control calvaria in the type of mineral produced (a lightly carbonated apatite), however we did observe increased mineral deposition in FGF2 treated calvaria. Raman imaging has great promise to detect the earliest mineral and matrix changes that occur in craniosynostosis.