Abstract:Nerve-sparing surgery is increasingly being applied to avoid functional deficits of the limbs and organs following surgery. Peripheral nerves that should be preserved are, however, sometimes misidentified due to similarity of shape and color to non-nerve tissues. To avoid misidentification of peripheral nerves, development of an in situ nerve detection method is desired. In this study, we report the label-free detection of ex vivo peripheral nerves of Wistar rats by using Raman spectroscopy. We obtained Raman … Show more
“…Spontaneous Raman microscopy provides quantitative information on substances according to their intrinsic, molecule-specific vibrational signatures of chemical bonds as a spectrum, which exhibits sharp spectral features based on specific molecular structures and conformations of tissues 10 11 12 13 . Recently, spontaneous Raman spectroscopy has been applied to evaluate various biological tissues or cells without chemical labeling 10 11 12 . For example, it can be used for cell imaging in the living organism 10 , and for discrimination of specific molecules in normal 12 or diseased tissues 13 14 .…”
Raman spectroscopy, which identifies intrinsic molecular constituents, has a potential for determining myocardial viability under label-free conditions. However, its suitability for evaluating myocardial ischaemia is undetermined. Focusing on cytochromes, i.e., representative molecules reflecting mitochondrial activity, we tested whether Raman spectroscopy is applicable for evaluating myocardial ischaemia especially during early ischaemic phase. We obtained spontaneous Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart upon 532-nm excitation before and during the “stopped-flow,” global ischaemia. Semi-quantitative values of the peak intensities at 750 and 1127 cm−1, which reflect reduced cytochromes c and b, increased immediately and progressively after induction of the stopped flow, indicating progressive reduction of the mitochondrial respiration. Such spectral changes emerged before the loss of 1) mitochondrial membrane potentials measured by the fluorescence intensity of tetramethyl rhodamine ethyl ester or 2) staining of the triphenyl tetrazolium chloride dye in the myocardium. The progressive increases in the Raman peaks by stopped flow were significantly retarded by ischaemic preconditioning. Sequential measurements of the peak intensities at 750 and 1127 cm−1 enabled early detection of the myocardial ischaemia based on the mitochondrial functions. These data suggest that Raman spectroscopy offers the potential to evaluate acute ischaemic heart under label-free conditions.
“…Spontaneous Raman microscopy provides quantitative information on substances according to their intrinsic, molecule-specific vibrational signatures of chemical bonds as a spectrum, which exhibits sharp spectral features based on specific molecular structures and conformations of tissues 10 11 12 13 . Recently, spontaneous Raman spectroscopy has been applied to evaluate various biological tissues or cells without chemical labeling 10 11 12 . For example, it can be used for cell imaging in the living organism 10 , and for discrimination of specific molecules in normal 12 or diseased tissues 13 14 .…”
Raman spectroscopy, which identifies intrinsic molecular constituents, has a potential for determining myocardial viability under label-free conditions. However, its suitability for evaluating myocardial ischaemia is undetermined. Focusing on cytochromes, i.e., representative molecules reflecting mitochondrial activity, we tested whether Raman spectroscopy is applicable for evaluating myocardial ischaemia especially during early ischaemic phase. We obtained spontaneous Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart upon 532-nm excitation before and during the “stopped-flow,” global ischaemia. Semi-quantitative values of the peak intensities at 750 and 1127 cm−1, which reflect reduced cytochromes c and b, increased immediately and progressively after induction of the stopped flow, indicating progressive reduction of the mitochondrial respiration. Such spectral changes emerged before the loss of 1) mitochondrial membrane potentials measured by the fluorescence intensity of tetramethyl rhodamine ethyl ester or 2) staining of the triphenyl tetrazolium chloride dye in the myocardium. The progressive increases in the Raman peaks by stopped flow were significantly retarded by ischaemic preconditioning. Sequential measurements of the peak intensities at 750 and 1127 cm−1 enabled early detection of the myocardial ischaemia based on the mitochondrial functions. These data suggest that Raman spectroscopy offers the potential to evaluate acute ischaemic heart under label-free conditions.
“…S2. Although the 2855 cm −1 band in previous studies 29, 30 is not seen here, it could be merged into the 2895 cm −1 band due to low spectral resolution of the instrument.Figure 1( a ) Bright-field image of a test sample containing bundles of peripheral nerve fibers (N), connective tissue (C), and a skeletal muscle tissue (M). The scale bar is 1 mm.…”
Raman spectroscopy allows label-free, minimally invasive, and accurate detection of peripheral nerves. However, the conventional Raman imaging technique is time-consuming when measuring a large area of a sample. Establishing a method for rapidly acquiring spatial distribution of a bundle of peripheral nerve fibers is an essential step for Raman spectroscopy towards application in clinical surgery. Here we present a multipoint Raman spectroscopic technique for rapid peripheral nerve imaging. In only 5 seconds, spectra at 32 points situated on ex vivo rat peripheral nerve bundles and adjoining connective tissues were acquired. Principal component regression and discriminant analysis of spectra revealed that the sensitivity, specificity and accuracy for nerve detection were 85.8%, 96.0%, and 90.8%, respectively. Of 158 peripheral nerves, 152 (96.2%) showed ratio of the number of nerve-positive prediction points to the total measurement points being 0.4 or larger, whereas 119 (99.2%) connective tissues among 120 showed ratio smaller than 0.4. Based on the ratio and a bright-field image of the sample, accurate visualization of peripheral nerves was implemented. The results indicated that the multipoint Raman spectroscopic technique is capable of rapid and accurate peripheral nerve imaging.
“…The wavelengths or wavenumbers of all of the spectra were calibrated by using the known Raman bands of ethanol excited by a 532‐nm laser. We employed a modified polynomial curve fitting method to separate the Raman and autofluorescence spectra . We estimated the autofluorescence superposed on the Raman spectra by calculating a modified least‐squares fifth‐order polynomial curve with 10 iterations and then subtracted this polynomial from the raw spectra.…”
Ukiyo‐e printing is a traditional Japanese multicolour woodblock printing style that flourished during the 18th century. Although this genre subsequently experienced a decline, interest in it has since re‐emerged. However, the materials and carving and printing technologies originally used to produce ukiyo‐e prints remain unclear due to a lack of technical information. In this study, we performed non‐invasive molecular imaging of the colour pigments in recently reproduced and original ukiyo‐e prints using Raman microspectroscopy to investigate ukiyo‐e production technologies. First, to confirm the effectiveness of this method, we obtained Raman spectral images of an ukiyo‐e print, Ohisa with a fan, that was recently reproduced using the current ukiyo‐e production protocols. The pigment species were easily identified based on their Raman spectra, and small pigment granules were observed along the paper fibres. Furthermore, we performed Raman microspectroscopy of an original ukiyo‐e print, Kabuki Actor Iwai Hanshirô VIII as Miuraya no Agemaki. Some pigment species were revealed by the Raman spectra. We also obtained high‐spatial‐resolution pigment distribution images and observed characteristic inhomogeneous colour pigment aggregations on and between the paper fibres depending on the reproduced and the original ukiyo‐e prints. Our results indicate that Raman microspectroscopy is a promising technique for determining the original ukiyo‐e print production technologies that are currently unknown.
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