Matthew V. Schulmerich

Transcutaneous fiber optic Raman spectroscopy of bone using annular illumination and a circular array of collection fibers

Transcutaneous bone Raman spectroscopy with an exciting annulus of 785-nm laser light surrounding the field of view of a circular array of collection fibers is demonstrated. The configuration provides distributed laser light. The annulus is located 2 to 3 mm beyond the edge of the field of view of the collection fibers to reject contributions from skin and other overlying tissues. Data are presented for rat and chicken tissue. For rat tibia, the carbonate/phosphate ratio measured at a depth of 1 mm below the skin is in error by 2.3% at an integration time of 120 s and within 10% at a 30-s integration time. For chicken tibia 4 mm below the skin surface, the error is less than 8% with a 120-s integration time.

Noninvasive Raman tomographic imaging of canine bone tissue.

Raman spectroscopic diffuse tomographic imaging has been demonstrated for the first time. It provides a noninvasive, label-free modality to image the chemical composition of human and animal tissue and other turbid media. This technique has been applied to image the composition of bone tissue within an intact section of a canine limb. Spatially distributed 785-nm laser excitation was employed to prevent thermal damage to the tissue. Diffuse emission tomography reconstruction was used, and the location that was recovered has been confirmed by micro-computed tomography (micro-CT) images.

Subsurface Raman Spectroscopy and Mapping Using a Globally Illuminated Non-Confocal Fiber-Optic Array Probe in the Presence of Raman Photon Migration

We report the use of a fiber-optic probe with global illumination and an array of 50 collection fibers (PhAT probe, Kaiser Optical Systems, Inc.) to obtain Raman spectra and 50 spatial element maps of polymers through overlayers of other polymers that are highly scattering. Band target entropy minimization (BTEM) is used to recover the spectra of the subsurface components and generate maps of their distributions. This approach to subsurface mapping is tested with model systems consisting of two or three layers of polyethylene, polytetrafluoroethylene (Teflon), and polyoxymethylene (Delrin) arranged in different geometries. Raman spectra and maps were obtained through overlayer thicknesses of up to 13 mm. Subsurface spatial resolution is achieved because each fiber views an asymmetric distribution of Raman scattered light from surface and subsurface components that depends on the position of the fiber relative to the depth and position of a component and the extent of photon diffusion through the system.

Transcutaneous Raman spectroscopy of murine bone in vivo.

Raman spectroscopy can provide valuable information about bone tissue composition in studies of bone development, biomechanics, and health. In order to study the Raman spectra of bone in vivo, instrumentation that enhances the recovery of subsurface spectra must be developed and validated. Five fiber-optic probe configurations were considered for transcutaneous bone Raman spectroscopy of small animals. Measurements were obtained from the tibia of sacrificed mice, and the bone Raman signal was recovered for each probe configuration. The configuration with the optimal combination of bone signal intensity, signal variance, and power distribution was then evaluated under in vivo conditions. Multiple in vivo transcutaneous measurements were obtained from the left tibia of 32 anesthetized mice. After collecting the transcutaneous Raman signal, exposed bone measurements were collected and used as a validation reference. Multivariate analysis was used to recover bone spectra from transcutaneous measurements. To assess the validity of the transcutaneous bone measurements cross-correlations were calculated between standardized spectra from the recovered bone signal and the exposed bone measurements. Additionally, the carbonate-to-phosphate height ratios of the recovered bone signals were compared to the reference exposed bone measurements. The mean cross-correlation coefficient between the recovered and exposed measurements was 0.96, and the carbonate-to-phosphate ratios did not differ significantly between the two sets of spectra (p > 0.05). During these first systematic in vivo Raman measurements, we discovered that probe alignment and animal coat color influenced the results and thus should be considered in future probe and study designs. Nevertheless, our noninvasive Raman spectroscopic probe accurately assessed bone tissue composition through the skin in live mice.

Subsurface and Transcutaneous Raman Spectroscopy and Mapping Using Concentric Illumination Rings and Collection with a Circular Fiber-Optic Array:

Different spatial separations between an illumination ring and a bundle of 50 collection fibers focused to collect light in the center of the ring were used to investigate the recovery of subsurface Raman spectra. The depth of Raman signal recovery and the preservation of spatial information in the recovered signal were investigated using polymer blocks stacked in different geometries. The illumination rings were then combined into a single data set to increase variation in the signal. Multivariate data analysis was used to recover the Raman spectra of the subsurface component. The Raman spectrum of a Delrin target was recoverable at depths up to 22.6 mm of overlying Teflon. Spatial information was lost at approximately 6.5 mm below the Teflon surface. The same protocols were used to recover canine bone spectra transcutaneously at depths up to 5 mm below the skins surface. The recovered bone spectra were validated by exposed bone measurements.

Optical clearing in transcutaneous Raman spectroscopy of murine cortical bone tissue

The effect of optical clearing with glycerol on the Raman spectra of bone tissue acquired transcutaneously on right and left tibiae from four mice is studied. Multiple transcutaneous measurements are obtained from each limb; glycerol is then applied as an optical clearing agent, and additional transcutaneous measurements are taken. Glycerol reduces the noise in the raw spectra (p=0.0037) and significantly improves the cross-correlation between the recovered bone factor and the exposed bone measurement in a low signal-to-noise region of the bone spectra (p=0.0245).

Image-guided Raman spectroscopic recovery of canine cortical bone contrast in situ

Raman scattering provides valuable biochemical and molecular markers for studying bone tissue composition with use in predicting fracture risk in osteoporosis. Raman tomography can image through a few centimeters of tissue but is limited by low spatial resolution. X-ray computed tomography (CT) imaging can provide high-resolution image-guidance of the Raman spectroscopic characterization, which enhances the quantitative recovery of the Raman signals, and this technique provides additional information to standard imaging methods. This hypothesis was tested in data measured from Teflon tissue phantoms and from a canine limb. Image-guided Raman spectroscopy (IG-RS) of the canine limb using CT images of the tissue to guide the recovery recovered a contrast of 145:1 between the cortical bone and background. Considerably less contrast was found without the CT image to guide recovery. This study presents the first known IG-RS results from tissue and indicates that intrinsically high contrasts (on the order of a hundred fold) are available.

Transcutaneous Raman spectroscopy of bone tissue using a non-confocal fiber optic array probe

We demonstrate the first transcutaneous Raman spectroscopic measurements of bone tissue employing a fiber optic probe with a uniformly illuminated array of collection fibers. Uniform illumination reduces local power density to avoid damage to specimens. Non-confocal operation provides efficient signal collection, and together with NIR laser excitation (785 nm diode laser) allows good depth penetration enabling recovery of spectra from beneath the skin. Multivariate data reduction is used to resolve Raman spectra of bone tissue from the spectra generated from overlying tissue. The probe utilizes non-confocal optics and uniform illumination allowing the system to collect spectra from above and below the range of best focus while applying a low power density. Despite extensive photon migration in the tissue specimens, the system can resolve transcutaneous signals because the collection cone of each fiber is asymmetric with respect to the center of illumination. Here we report preliminary results of tissue specimens taken from chicken tibia as well as from a human elbow.

Transcutaneous Raman spectroscopy of bone global sampling and ring/disk fiber optic probes

We have used fiber optic probes with global illumination/collection (PhAT probe, Kaiser Optical Systems) and ring illumination/disk collection configurations for transcutaneous Raman spectroscopy of bone tissue. Both illumination/collection schemes can be used for recovery of spectra of subsurface components. In this paper the global illumination configuration provides minimum local power density and so minimizes the probability of damage to specimens, animals or human subjects. It also allows non-destructive subsurface mapping under certain conditions. The ring/disk probe utilizes a ring of laser light and collects Raman scatter from within the diameter of the ring. This design distributes the laser power for efficient heat dissipation and provides a better collection ratio of subsurface to surface components than the global illumination design. For non-invasive tissue spectroscopy the ring/disk design also provides better rejection of fluorescence from melanocytes. We have tested the performance of these Raman probes on polymer model systems and chicken tibiae.

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.