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.
Background fluorescence can often complicate the use of Raman microspectroscopy in the study of musculoskeletal tissues. Such fluorescence interferences are undesirable as the Raman spectra of matrix and mineral phases can be used to differentiate between normal and pathological or microdamaged bone. Photobleaching with the excitation laser provides a non-invasive method for reducing background fluorescence, enabling 532 nm Raman hyperspectral imaging
of bone tissue. The signal acquisition time for a 400 point Raman line image is reduced to 1-4 seconds using electronmultiplying
CCD (EMCCD) detector, enabling acquisition of Raman images in less than 10 minutes. Rapid photobleaching depends upon multiple scattering effects in the tissue specimen and is applicable to some, but not all experimental situations.
Tissue modulated Raman spectroscopy was used noninvasively to measure blood glucose concentration in people with type I and type II diabetes with HemoCue fingerstick measurements being used as reference. Including all of the 49 measurements, a Clarke error grid analysis of the noninvasive measurements showed that 72% were A range, i.e., clinically accurate, 20% were B range, i.e., clinically benign, with the remaining 8% of measurements being essentially erroneous, i.e., C, D, or E range. Rejection of 11 outliers gave a correlation coefficient of 0.80, a standard deviation of 22 mg/dL with p<0.0001 for N=38 and places all but one of the measurements in the A and B ranges. The distribution of deviations of the noninvasive glucose measurements from the fingerstick glucose measurements is consistent with the suggestion that there are at least two systematic components in addition to the random noise associated with shot noise, charge coupled device spiking, and human factors. One component is consistent with the known variation of fingerstick glucose concentration measurements from laboratory reference measurements made using plasma or whole blood. A weak but significant correlation between the deviations of noninvasive measurements from fingerstick glucose measurements and the test subject's hemoglobin concentration was also observed.
Raman spectroscopy is used as a probe of ultrastructural (molecular) changes in both the mineral and matrix (protein and glycoprotein, predominantly type I collagen) components of murine cortical bone as it responds to loading in the elastic regime. At the ultrastructural level, crystal structure and protein secondary structure distort as the tissue is loaded. These structural changes are followed as perturbations to tissue spectra. We load tissue in a custom-made dynamic mechanical tester that fits on the stage of a Raman microprobe and can accept hydrated tissue specimens. As the specimen is loaded in tension and/or compression, the shifts in mineral P-O4v1 and relative band heights in the Amide III band envelope are followed with the microprobe. Average load is measured using a load cell while the tissue is loaded under displacement control. Changes occur in both the mineral and matrix components of bone as a response to elastic deformation. We propose that the mineral apatitic crystal lattice is deformed by movement of calcium and other ions. The matrix is proposed to respond by deformation of the collagen backbone. Raman microspectroscopy shows that bone mineral is not a passive contributor to tissue strength. The mineral active response to loading may function as a local energy storage and dissipation mechanism, thus helping to protect tissue from catastrophic damage.
We present tissue modulated Raman spectroscopy as a technique for noninvasively measuring the concentration of blood analytes in vivo. We present preliminary data used to determine the best methods for analyzing our data. These experiments provide additional proof that we are indeed able to obtain the spectra of human blood in vivo and noninvasively. We discuss differences between our spectra and spectra of bulk blood in vitro. We also discuss the variations between individuals and the impact of those variations on our noninvasive blood glucose measurements.
We have recently presented the first Raman spectra of in vivo human blood. A brief review of how to obtain such spectra and normalize them to the appropriate blood volume is given showing how to produce spectra that can be used for noninvasive quantitative analysis of blood in vivo. New clinical data from individuals and groups completely reproduce and extend all the earlier results. These new data reveal how certain small differences between individuals result in some variability in their noninvasive quantitation. We show the origin of this variability and how to obtain quantitative corrections based entirely on the individual measurement and tabulated data.
We recently presented the first Raman spectra of in vivo human blood. A brief review of how to obtain such spectra and then normalize them to the appropriate blood volume is given showing how to produce spectra that can be used for noninvasive quantitative analysis of blood in vivo. A more careful comparison of tissue modulated spectra with static in vitro and invasive in vivo spectra suggests that there are small microcirculation differences between individuals resulting in some variability in their noninvasive quantitation. This variability is based on the mechanism for blood volume normalization and various means for obtaining necessary corrections are suggested. We present new clinical data from individuals and groups supporting this mechanism and suggesting how such measurements might also be used to quantify various microcirculation abnormalities.
KEYWORDS: Raman spectroscopy, Tissues, In vivo imaging, Skin, Spectroscopy, In vitro testing, Proteins, Near infrared spectroscopy, Modulation, Near infrared
We report the use of near infrared vibrational spectroscopy to noninvasively probe the in-vivo lipid and aqueous phases of skin and near surface tissues under conditions of thermal and chemical modulation. We demonstrate thermally induced order- disorder transitions in lipids that can be directly compared to well known behavior of in-vitro samples of phospholipid bilayers and bulk fatty acids. We show reversible chemical modification of aqueous phase proteins which are also directly comparable to well known phenomena involving in-vitro proteins. The results of these studies demonstrate the capacity for noninvasively probing live human tissues on the molecular level using near infrared vibrational spectroscopy. This capacity suggests numerous potential applications ranging from assessing the efficacy of cosmetics, skin care treatments and transdermal therapeutic agents/treatments to serving as a diagnostic of various skin ailments, e.g. melanoma.
We report the first noninvasive Raman spectra of in vivo human blood. 'Tissue modulation' involves the use of thermal and/or mechanical stimulus to produce particular spatiotemporal distributions of mobile tissues, i.e. capillary blood, among nonmobile tissues, i.e. epidermis. Using this approach we have obtained three mutually independent lines of evidence, which unequivocally associate Raman spectra we have obtained with human blood. These spectral compare well with published spectra from other researchers of in vitro human blood. The results of a recent clinical study comparing our noninvasive in vivo spectroscopic measurements with simultaneous conventional in vitro measurements clearly demonstrate the efficacy of the tissue modulation approach. These results will be discussed in the context of noninvasive monitoring of a variety of analytes, i.e. glucose.
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