RESUMO
Erythropoietic protoporphyria (EPP) is a rare inherited disorder of porphyrin metabolism that can cause liver damage and cholestatic hepatocellular failure. We report a case of EPP in a teenaged male who underwent liver biopsy for investigation of liver dysfunction of unknown cause. The diagnosis was not made until a re-biopsy approximately three years later, when the patient presented with recurrent skin lesions and elevated blood and urinary protoporphyrin levels. The liver biopsies contained brownish deposits that exhibited birefringence under polarized light and porphyrin fluorescence under fluorescence spectroscopy. EPP should be considered in young patients with unexplained liver dysfunction, skin symptoms, and seasonal changes in symptoms. Fluorescence spectroscopy of liver biopsy tissue can be a useful tool in the diagnosis of EPP.
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Non-alcoholic fatty liver disease (NAFLD) is associated with the chronic progression of fibrosis. In general, the progression of liver fibrosis is determined by a histopathological assessment with a collagen-stained section; however, the ultra-early stage of liver fibrosis is challenging to identify because of the low sensitivity in the collagen-selective staining method. In the present study, we demonstrate the feasibility of second-harmonic generation (SHG) microscopy in the histopathological diagnosis of the liver of NAFLD patients for the quantitative assessment of the ultra-early stage of fibrosis. We investigated four representative NAFLD patients with early stages of fibrosis. SHG microscopy visualised well-matured fibrotic structures and early fibrosis diffusely involving liver tissues, whereas early fibrosis is challenging to be identified by conventional histopathological methods. Furthermore, the SHG emission directionality analysis revealed the maturation of each collagen fibre of each patient. As a result, SHG microscopy is feasible for assessing liver fibrosis on NAFLD patients, including the ultra-early stage of liver fibrosis that is difficult to diagnose by the conventional histopathological method. The assessment method of the ultra-early fibrosis by using SHG microscopy may serve as a crucial means for pathological, clinical, and prognostic diagnosis of NAFLD patients.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Microscopia de Geração do Segundo Harmônico , Biópsia/métodos , Colágeno , Fibrose , Humanos , Fígado/diagnóstico por imagem , Fígado/patologia , Cirrose Hepática/diagnóstico por imagem , Cirrose Hepática/etiologia , Hepatopatia Gordurosa não Alcoólica/complicaçõesRESUMO
5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is widely used for the intraoperative detection of malignant tumors. However, the fluorescence emission profiles of the accompanying necrotic regions of these tumors have yet to be determined. To address this, we performed fluorescence and high-performance liquid chromatography (HPLC) analyses of necrotic tissues of squamous cancer after 5-ALA administration. In resected human lymph nodes of metastatic squamous cell carcinoma, we found a fluorescence peak at approximately 620 nm in necrotic lesions, which was distinct from the PpIX fluorescence peak at 635 nm for viable cancer lesions. Necrotic lesions obtained from a subcutaneous xenograft model of human B88 oral squamous cancer also emitted the characteristic fluorescence peak at 620 nm after light irradiation: the fluorescence intensity ratio (620 nm/635 nm) increased with the energy of the irradiation light. HPLC analysis revealed a high content ratio of uroporphyrin I (UPI)/total porphyrins in the necrotic cores of murine tumors, indicating that UPI is responsible for the 620 nm peak. UPI accumulation in necrotic tissues after 5-ALA administration was possibly due to the failure of the heme biosynthetic pathway. Taken together, fluorescence imaging of UPI after 5-ALA administration may be applicable for the evaluation of tumor necrosis.
Assuntos
Ácido Aminolevulínico/administração & dosagem , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patologia , Uroporfirinas/metabolismo , Idoso , Ácido Aminolevulínico/uso terapêutico , Animais , Carcinoma de Células Escamosas/tratamento farmacológico , Linhagem Celular Tumoral , Cromatografia Líquida de Alta Pressão , Modelos Animais de Doenças , Neoplasias Esofágicas/tratamento farmacológico , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Feminino , Humanos , Masculino , Camundongos , Pessoa de Meia-Idade , Modelos Biológicos , Necrose , Espectrometria de FluorescênciaRESUMO
Inactivation technology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is certainly a critical measure to mitigate the spread of coronavirus disease 2019 (COVID-19). A deep ultraviolet light-emitting diode (DUV-LED) would be a promising candidate to inactivate SARS-CoV-2, based on the well-known antiviral effects of DUV on microorganisms and viruses. However, due to variations in the inactivation effects across different viruses, quantitative evaluations of the inactivation profile of SARS-CoV-2 by DUV-LED irradiation need to be performed. In the present study, we quantify the irradiation dose of DUV-LED necessary to inactivate SARS-CoV-2. For this purpose, we determined the culture media suitable for the irradiation of SARS-CoV-2 and optimized the irradiation apparatus using commercially available DUV-LEDs that operate at a center wavelength of 265, 280, or 300 nm. Under these conditions, we successfully analyzed the relationship between SARS-CoV-2 infectivity and the irradiation dose of the DUV-LEDs at each wavelength without irrelevant biological effects. In conclusion, total doses of 1.8 mJ/cm2 for 265 nm, 3.0 mJ/cm2 for 280 nm, and 23 mJ/cm2 for 300 nm are required to inactivate 99.9% of SARS-CoV-2. Our results provide quantitative antiviral effects of DUV irradiation on SARS-CoV-2, serving as basic knowledge of inactivation technologies against SARS-CoV-2.
Assuntos
SARS-CoV-2/efeitos da radiação , Terapia Ultravioleta/métodos , Inativação de Vírus/efeitos da radiação , COVID-19/epidemiologia , COVID-19/transmissão , Humanos , SARS-CoV-2/metabolismo , Raios Ultravioleta , Viroses/prevenção & controleRESUMO
We have synthesized a cyan fluorescent boron complex based on a tridentate imidazo[1,5-a]pyridine ligand. The boron complex was found to have potential applications as not only a chiroptical material but also a heavy-atom-free mitochondria-targeted photosensitizer for cancer treatment.
RESUMO
We have synthesized a series of quaternized imidazo[1,2-a]pyridines in three steps from commercially available reagents. These compounds exhibit blue fluorescence emission at around 425 nm with good quantum yields. In addition, one specific compound was found to work as not only a two- and three-photon excitable mitochondria imaging agent, but also a therapeutic agent upon continuous irradiation conditions.
Assuntos
PiridinasRESUMO
The preparation of a hierarchically assembled Ag nanostructures based on a nanocrystalline assembly was demonstrated using an Ag(I) complex of a dipeptide (AspDap). By heating under N2 gas, a spherical assembly of a nanocrystalline dipeptide-Ag(I) complex (diameter 4-5â µm), which has a morphology similar to the assembled structure of the dipeptide, was transformed to an assembly of Ag nanostructures, where the micrometre-order crystalline morphology was maintained. In addition, detailed scanning electron microscopy studies revealed that Ag nanoparticles (diameter ca.â 10â nm) were formed on the surface of the Ag nanostructure. When the Ag(I) ions were reduced to Ag(0), this phenomenon exhibited surface dependence due to the anisotropic two-dimensional Ag(I) arrangement in the crystals. Thermogravimetric measurements and X-ray photoelectron spectroscopy revealed that the reduction proceeds in a stepwise manner around 200-250 °C, together with the removal of primary and secondary carboxylic groups in the dipeptide. Comparison with the heating process of the crystalline Ag(I) complex of ß-alanine indicated that stepwise reduction is key for maintaining the original micrometre-order morphology.
Assuntos
Complexos de Coordenação/química , Nanopartículas Metálicas/química , Peptídeos/química , Prata/química , Complexos de Coordenação/síntese química , Oxirredução , Peptídeos/síntese químicaRESUMO
Understanding the viability of the ischemic myocardial tissue is a critical issue in determining the appropriate surgical procedure for patients with chronic heart failure after myocardial infarction (MI). Conventional MI evaluation methods are; however, preoperatively performed and/or give an indirect information of myocardial viability such as shape, color, and blood flow. In this study, we realize the evaluation of MI in patients undergoing cardiac surgery by Raman spectroscopy under label-free conditions, which is based on intrinsic molecular constituents related to myocardial viability. We identify key signatures of Raman spectra for the evaluation of myocardial viability by evaluating the infarct border zone myocardium that were excised from five patients under surgical ventricular restoration. We also obtain a prediction model to differentiate the infarcted myocardium from the non-infarcted myocardium by applying partial least squares regression-discriminant analysis (PLS-DA) to the Raman spectra. Our prediction model enables identification of the infarcted tissues and the non-infarcted tissues with sensitivities of 99.98% and 99.92%, respectively. Furthermore, the prediction model of the Raman images of the infarct border zone enabled us to visualize boundaries between these distinct regions. Our novel application of Raman spectroscopy to the human heart would be a useful means for the detection of myocardial viability during surgery.
Assuntos
Cardiomiopatias/diagnóstico por imagem , Ventrículos do Coração/diagnóstico por imagem , Infarto do Miocárdio/diagnóstico por imagem , Miocárdio/patologia , Análise Espectral Raman/métodos , Procedimentos Cirúrgicos Cardíacos , Cardiomiopatias/patologia , Cardiomiopatias/cirurgia , Diagnóstico Diferencial , Estudos de Viabilidade , Ventrículos do Coração/patologia , Ventrículos do Coração/cirurgia , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Modelos Cardiovasculares , Infarto do Miocárdio/patologia , Infarto do Miocárdio/cirurgia , Valor Preditivo dos Testes , Prognóstico , Procedimentos de Cirurgia Plástica , Sensibilidade e Especificidade , SoftwareRESUMO
Optical frequency combs (OFCs) have attracted attention as optical frequency rulers due to their tooth-like discrete spectra together with their inherent mode-locking nature and phase-locking control to a frequency standard. Based on this concept, their applications until now have been demonstrated in the fields of optical frequency metrology. However, if the utility of OFCs can be further expanded beyond their application by exploiting new aspects of OFCs, this will lead to new developments in optical metrology and instrumentation. Here, we report a fiber sensing application of OFCs based on a coherent link between the optical and radio frequencies, enabling high-precision refractive index measurement based on frequency measurement in radio-frequency (RF) region. Our technique encodes a refractive index change of a liquid sample into a repetition frequency of OFC by a combination of an intracavity multi-mode-interference fiber sensor and wavelength dispersion of a cavity fiber. Then, the change in refractive index is read out by measuring the repetition frequency in RF region based on a frequency standard. Use of an OFC as a photonic RF converter will lead to the development of new applications in high-precision fiber sensing with the help of functional fiber sensors and precise RF measurement.
RESUMO
We propose an optical frequency comb (OFC)-based strain sensing method, namely OFC sensing cavity, which is capable of radio-frequency (RF)-based strain measurement. We developed a null-method-based strain sensing system with a comb-spacing-stabilized OFC generator. We realized strain measurement from 1.83 µÎµ to 1800 µÎµ with a sensing fiber length of 20 mm. The measurable strain frequency range of the developed strain sensing system was from 0 to 310 Hz. Owing to the use of RF-based strain measurement, our approach would be a useful and powerful tool for sensing of strain or other physical quantities, and the concept of the OFC sensing cavity is a new aspect of OFC technology.
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Protoporphyrin IX (PpIX), a biochemical converted from 5-aminolevulinc acid (5-ALA) in living cells, is useful for intraoperative fluorescent detection of cancer metastasis in lymph nodes (LNs). However, unknown is whether the fluorescence of PpIX can be detected in the LNs when they coexist with indigo carmine, a blue dye commonly used for identification of sentinel LNs during surgery. To address this issue, we sought to evaluate the diagnostic usefulness of PpIX fluorescence in the presence of indigo carmine in a mouse LN metastasis model of rectal cancer after administration of 5-ALA. Spectral analysis of pure chemicals revealed that the absorption spectrum of indigo carmine widely overlapped with the fluorescence spectrum of PpIX specifically at the peak of 632nm, a common emission wavelength for detecting PpIX, but not at the other peak of 700nm. Due to such spectral overlap, the PpIX fluorescence intensity was significantly attenuated by mixture with indigo carmine at 632nm, but not at 700nm. Accordingly, fluorescent measurements of the mouse metastatic LN revealed more intense presentation of PpIX at 700nm than at 632nm, indicating that the diagnostic usefulness is greater at 700nm than at 632nm for the indigo carmine-dyed LNs after administration of 5-ALA. From these observations, we propose that the fluorescence measurement is more efficient at 700nm than at 632nm for detection of PpIX in metastatic LNs stained with indigo carmine.
Assuntos
Neoplasias Colorretais/patologia , Corantes/farmacologia , Índigo Carmim/farmacologia , Linfonodos/diagnóstico por imagem , Imagem Óptica/métodos , Protoporfirinas/farmacologia , Animais , Corantes/farmacocinética , Índigo Carmim/farmacocinética , Camundongos , Metástase Neoplásica , Protoporfirinas/farmacocinéticaRESUMO
A single, free-running, dual-wavelength mode-locked, erbium-doped fibre laser was exploited to measure the absolute frequency of continuous-wave terahertz (CW-THz) radiation in real time using dual THz combs of photo-carriers (dual PC-THz combs). Two independent mode-locked laser beams with different wavelengths and different repetition frequencies were generated from this laser and were used to generate dual PC-THz combs having different frequency spacings in photoconductive antennae. Based on the dual PC-THz combs, the absolute frequency of CW-THz radiation was determined with a relative precision of 1.2 × 10-9 and a relative accuracy of 1.4 × 10-9 at a sampling rate of 100 Hz. Real-time determination of the absolute frequency of CW-THz radiation varying over a few tens of GHz was also demonstrated. Use of a single dual-wavelength mode-locked fibre laser, in place of dual mode-locked lasers, greatly reduced the size, complexity, and cost of the measurement system while maintaining the real-time capability and high measurement precision.
RESUMO
Terahertz (THz) spectroscopy is a promising method for analysing polar gas molecules mixed with unwanted aerosols due to its ability to obtain spectral fingerprints of rotational transition and immunity to aerosol scattering. In this article, dynamic THz spectroscopy of acetonitrile (CH3CN) gas was performed in the presence of smoke under the atmospheric pressure using a fibre-based, asynchronous-optical-sampling THz time-domain spectrometer. To match THz spectral signatures of gas molecules at atmospheric pressure, the spectral resolution was optimized to 1 GHz with a measurement rate of 1 Hz. The spectral overlapping of closely packed absorption lines significantly boosted the detection limit to 200 ppm when considering all the spectral contributions of the numerous absorption lines from 0.2 THz to 1 THz. Temporal changes of the CH3CN gas concentration were monitored under the smoky condition at the atmospheric pressure during volatilization of CH3CN droplets and the following diffusion of the volatilized CH3CN gas without the influence of scattering or absorption by the smoke. This system will be a powerful tool for real-time monitoring of target gases in practical applications of gas analysis in the atmospheric pressure, such as combustion processes or fire accident.
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5-aminolevulinic acid (5-ALA)-based fluorescence diagnosis is now clinically applied for accurate and ultrarapid diagnosis of malignant lesions such as lymph node metastasis during surgery. 5-ALA-based diagnosis evaluates fluorescence intensity of a fluorescent metabolite of 5-ALA, protoporphyrin IX (PPIX); however, the fluorescence of PPIX is often affected by autofluorescence of tissue chromophores, such as collagen and flavins. In this study, we demonstrated PPIX fluorescence estimation with autofluorescence elimination for 5-ALA-based fluorescence diagnosis of malignant lesions by simplified and optimized multispectral imaging. We computationally optimized observation wavelength regions for the estimation of PPIX fluorescence in terms of minimizing prediction error of PPIX fluorescence intensity in the presence of typical chromophores, collagen and flavins. By using the fluorescence intensities of the optimized wavelength regions, we verified quantitative detection of PPIX fluorescence by using chemical mixtures of PPIX, flavins, and collagen. Furthermore, we demonstrated detection capability by using metastatic and non-metastatic lymph nodes of colorectal cancer patients. These results suggest the potential and usefulness of the background-free estimation method of PPIX fluorescence for 5-ALA-based fluorescence diagnosis of malignant lesions, and we expect this method to be beneficial for intraoperative and rapid cancer diagnosis.
Assuntos
Ácido Aminolevulínico/metabolismo , Neoplasias Colorretais/diagnóstico por imagem , Corantes Fluorescentes/metabolismo , Processamento de Imagem Assistida por Computador/métodos , Linfonodos/diagnóstico por imagem , Protoporfirinas/química , Ácido Aminolevulínico/administração & dosagem , Biotransformação , Linhagem Celular Tumoral , Colágeno/química , Neoplasias Colorretais/patologia , Neoplasias Colorretais/cirurgia , Flavinas/química , Corantes Fluorescentes/administração & dosagem , Humanos , Excisão de Linfonodo , Linfonodos/patologia , Linfonodos/cirurgia , Metástase Linfática , Imagem Óptica , Protoporfirinas/metabolismo , Razão Sinal-Ruído , SoftwareRESUMO
Photodynamic diagnosis based on 5-aminolevulinic acid-induced protoporphyrin IX has been clinically applied in many fields based upon its evidenced efficacy and adequate safety. In order to establish a personalized medicine approach for treating gastric cancer patients, rapid intraoperative detection of malignant lesions has become important. Feasibility of photodynamic diagnosis using 5-aminolevulinic acid for gastric cancer patients has been investigated, especially for the detection of peritoneal dissemination and lymph node metastasis. This method enables intraoperative real-time fluorescence detection of peritoneal dissemination, exhibiting higher sensitivity than white light observation without histopathological examination. The method also enables detection of metastatic foci within excised lymph nodes, exhibiting a diagnostic accuracy comparable to that of a current molecular diagnostics technique. Although several complicating issues still need to be resolved, such as the effect of tissue autofluorescence and the insufficient depth penetration of excitation light, this simple and rapid method has the potential to become a useful diagnostic tool for gastric cancer, as well as urinary bladder cancer and glioma.
Assuntos
Ácido Aminolevulínico/administração & dosagem , Biomarcadores Tumorais/metabolismo , Cuidados Intraoperatórios/métodos , Técnicas de Diagnóstico Molecular , Neoplasias Peritoneais/diagnóstico , Protoporfirinas/metabolismo , Neoplasias Gástricas/diagnóstico , Animais , Humanos , Medições Luminescentes , Metástase Linfática , Neoplasias Peritoneais/metabolismo , Neoplasias Peritoneais/secundário , Neoplasias Peritoneais/cirurgia , Valor Preditivo dos Testes , Reprodutibilidade dos Testes , Neoplasias Gástricas/metabolismo , Neoplasias Gástricas/patologia , Neoplasias Gástricas/cirurgiaRESUMO
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 spectra of peripheral nerves (myelinated and unmyelinated nerves) and their adjacent tissues of Wistar rats without any treatment such as fixation and/or staining. For the identification of tissue species and further analysis of spectral features, we proposed a principal component regression-based discriminant analysis with representative Raman spectra of peripheral nerves and their adjacent tissues. Our prediction model selectively detected myelinated nerves and unmyelinated nerves of Wistar rats with respective sensitivities of 95.5% and 88.3% and specificities of 99.4% and 93.5%. Furthermore, important spectral features for the identification of tissue species were revealed by detailed analysis of principal components of representative Raman spectra of tissues. Our proposed approach may provide a unique and powerful tool for peripheral nerve detection for nerve-sparing surgery in the future.
Assuntos
Nervos Periféricos/química , Análise Espectral Raman , Tecido Adiposo/química , Tecido Adiposo/patologia , Animais , Análise Discriminante , Masculino , Músculo Esquelético/química , Músculo Esquelético/patologia , Fibras Nervosas Mielinizadas/química , Fibras Nervosas Mielinizadas/patologia , Nervos Periféricos/patologia , Análise de Componente Principal , Ratos , Ratos WistarRESUMO
The absorption spectrum of light is known to be a "molecular fingerprint" that enables analysis of the molecular type and its amount. It would be useful to measure the absorption spectrum in single cell in order to investigate the cellular status. However, cells are too thin for their absorption spectrum to be measured. In this study, we developed an optical-cavity-enhanced absorption spectroscopic microscopy method for two-dimensional absorption imaging. The light absorption is enhanced by an optical cavity system, which allows the detection of the absorption spectrum with samples having an optical path length as small as 10 µm, at a subcellular spatial resolution. Principal component analysis of various types of cultured mammalian cells indicates absorption-based cellular diversity. Interestingly, this diversity is observed among not only different species but also identical cell types. Furthermore, this microscopy technique allows us to observe frozen sections of tissue samples without any staining and is capable of label-free biopsy. Thus, our microscopy method opens the door for imaging the absorption spectra of biological samples and thereby detecting the individuality of cells.
Assuntos
Microscopia/métodos , Análise de Célula Única/métodos , Animais , Células COS , Chlorocebus aethiops , Células HeLa , Humanos , Luz , Camundongos , Células NIH 3T3 , Células PC12 , Análise de Componente Principal , RatosRESUMO
Accurate evaluation of metastatic lymph nodes (LNs) is indispensable for adequate treatment of colorectal cancer (CRC) patients. Here, we demonstrate detection of metastases of human CRC in removed fresh LNs using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence. A spectral unmixing method was employed to reduce the overlap of collagen autofluorescence on PpIX fluorescence. A total of 17 surgery patients with advanced CRC were included in this study. After 5-ALA at a dose of 15 mg/kg of body weight was applied orally 2 h prior to surgery, 87 LNs were subjected to spectral fluorescence imaging and histopathological diagnosis, and statistical analysis was performed. No apparent side effect was observed to be associated with 5-ALA administration. The spectral unmixing fluorescence intensity of PpIX in metastatic LNs was 10.2-fold greater than that in nonmetastaic LNs. The receiver-operating-characteristic (ROC) analysis showed that the area under the curve (AUC) was calculated as 0.95. Our results show the potential of 5-ALA-induced PpIX fluorescence processed by spectral unmixing for detecting metastases in excised fresh LNs from patients with CRC, suggesting that this rapid and feasible method is applicable to gross evaluation of resected LN samples in pathology laboratories.
Assuntos
Ácido Aminolevulínico , Neoplasias Colorretais/cirurgia , Metástase Linfática/diagnóstico , Protoporfirinas , Adulto , Idoso , Idoso de 80 Anos ou mais , Ácido Aminolevulínico/química , Ácido Aminolevulínico/farmacologia , Neoplasias Colorretais/diagnóstico , Neoplasias Colorretais/patologia , Feminino , Fluorescência , Humanos , Linfonodos/patologia , Linfonodos/cirurgia , Metástase Linfática/patologia , Masculino , Pessoa de Meia-Idade , Protoporfirinas/químicaRESUMO
Detection of peripheral nerve tissues during surgery is required to avoid neural disturbance following surgery as an aspect of realizing better functional outcome. We provide a proof-of-principle demonstration of a label-free detection technique of peripheral nerve tissues, including myelinated and unmyelinated nerves, against adjacent tissues that employ spontaneous Raman microspectroscopy. To investigate the Raman spectral features of peripheral nerves in detail, we used unfixed sectioned samples. Raman spectra of myelinated nerve, unmyelinated nerve, fibrous connective tissue, skeletal muscle, tunica media of blood vessel, and adipose tissue of Wistar rats were analyzed, and Raman images of the tissue distribution were constructed using the map of the ordinary least squares regression (OLSR) estimates. We found that nerve tissues exhibited a specific Raman spectrum arising from axon or myelin sheath, and that the nerve tissues can be selectively detected against the other tissues. Moreover, myelinated and unmyelinated nerves can be distinguished by the intensity differences of 2,855 cm⻹, and 2,945 cm⻹, which are mainly derived from lipid and protein contents of nerve fibers. We applied this method to unfixed section samples of human periprostatic tissues excised from prostatic cancer patients. Myelinated nerves, unmyelinated nerves, fibrous connective tissues, and adipose tissues of the periprostatic tissues were separately detected by OLSR analysis. These results suggest the potential of the Raman spectroscopic observation for noninvasive and label-free nerve detection, and we expect this method could be a key technique for nerve-sparing surgery.