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      A handheld laser scanning confocal reflectance imaging–confocal Raman microspectroscopy system

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          Abstract

          Confocal reflectance microscopy and confocal Raman spectroscopy have shown potential for non-destructive analysis of samples at micron-scale resolutions. Current studies utilizing these techniques often employ large bench-top microscopes, and are not suited for use outside of laboratory settings. We have developed a microscope which combines laser scanning confocal reflectance imaging and confocal Raman spectroscopy into a compact handheld probe that is capable of high-resolution imaging and spectroscopy in a variety of settings. The compact size of the probe is largely due to the use of a MEMS mirror for beam scanning. The probe is capable of axial resolutions of up to 4 μm for the confocal imaging channel and 10 μm for the confocal Raman spectroscopy channel. Here, we report instrument design, characterize optical performance, and provide images and spectra from normal skin to demonstrate the instrument’s capabilities for clinical diagnostics.

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          Most cited references19

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          Dental microwear texture analysis shows within-species diet variability in fossil hominins.

          Reconstructing the diets of extinct hominins is essential to understanding the paleobiology and evolutionary history of our lineage. Dental microwear, the study of microscopic tooth-wear resulting from use, provides direct evidence of what an individual ate in the past. Unfortunately, established methods of studying microwear are plagued with low repeatability and high observer error. Here we apply an objective, repeatable approach for studying three-dimensional microwear surface texture to extinct South African hominins. Scanning confocal microscopy together with scale-sensitive fractal analysis are used to characterize the complexity and anisotropy of microwear. Results for living primates show that this approach can distinguish among diets characterized by different fracture properties. When applied to hominins, microwear texture analysis indicates that Australopithecus africanus microwear is more anisotropic, but also more variable in anisotropy than Paranthropus robustus. This latter species has more complex microwear textures, but is also more variable in complexity than A. africanus. This suggests that A. africanus ate more tough foods and P. robustus consumed more hard and brittle items, but that both had variable and overlapping diets.
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            In vivo confocal scanning laser microscopy of human skin II: advances in instrumentation and comparison with histology.

            In 1995, we reported the construction of a video-rate scanning laser confocal microscope for imaging human skin in vivo. Since then, we have improved the resolution, contrast, depth of imaging, and field of view. Confocal images of human skin are shown with experimentally measured lateral resolution 0.5-1.0 microm and axial resolution (section thickness) 3-5 microm at near-infrared wavelengths of 830 nm and 1064 nm; this resolution compares well to that of histology which is based on typically 5 microm thin sections. Imaging is possible to maximum depth of 350 microm over field of view of 160-800 microm. A mechanical skin-contact device was developed to laterally stabilize the imaging site to within +/- 25 microm in the presence of subject motion. Based on these results, we built a small, portable, and robust confocal microscope that is capable of imaging normal and abnormal skin morphology and dynamic processes in vivo, in both laboratory and clinical settings. We report advances in confocal microscope instrumentation and methods, an optimum range of parameters, improved images of normal human skin, and comparison of confocal images with histology.
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              Automated method for subtraction of fluorescence from biological Raman spectra.

              One of the challenges of using Raman spectroscopy for biological applications is the inherent fluorescence generated by many biological molecules that underlies the measured spectra. This fluorescence can sometimes be several orders of magnitude more intense than the weak Raman scatter, and its presence must be minimized in order to resolve and analyze the Raman spectrum. Several techniques involving hardware and software have been devised for this purpose; these include the use of wavelength shifting, time gating, frequency-domain filtering, first- and second-order derivatives, and simple curve fitting of the broadband variation with a high-order polynomial. Of these, polynomial fitting has been found to be a simple but effective method. However, this technique typically requires user intervention and thus is time consuming and prone to variability. An automated method for fluorescence subtraction, based on a modification to least-squares polynomial curve fitting, is described. Results indicate that the presented automated method is proficient in fluorescence subtraction, repeatability, and in retention of Raman spectral lineshapes.
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                Author and article information

                Journal
                Biomed Opt Express
                BOE
                Biomedical Optics Express
                Optical Society of America
                2156-7085
                09 February 2012
                01 March 2012
                09 February 2012
                : 3
                : 3
                : 488-502
                Affiliations
                [1 ]Department of Biomedical Engineering, Station B, Box 351631, Vanderbilt University, Nashville TN, 37235, USA
                [2 ]Department of Electrical and Computer Engineering, Montana State University, 610 Cobleigh Hall, Bozeman, MT, 59717, USA
                Author notes
                Article
                157165
                10.1364/BOE.3.000488
                3296537
                22435097
                c02e983f-9642-4f07-9545-e4da410ba24f
                ©2012 Optical Society of America

                This is an open-access article distributed under the terms of the Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 Unported License, which permits download and redistribution, provided that the original work is properly cited. This license restricts the article from being modified or used commercially.

                History
                : 27 October 2011
                : 13 January 2012
                : 14 January 2012
                Funding
                Funded by: National Institutes of Health, National Cancer Institute
                Award ID: R01-CA-1774461
                Categories
                Clinical Instrumentation
                Custom metadata
                True
                0

                Vision sciences
                (170.3890) medical optics instrumentation,(170.1790) confocal microscopy,(170.5660) raman spectroscopy,(170.6935) tissue characterization,(170.1870) dermatology,(170.1610) clinical applications

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