RESUMEN
Due to the shortage of healthy donor organs, steatotic livers are commonly used for transplantation, placing patients at higher risk for graft dysfunction and lower survival rates. Raman Spectroscopy is a technique which has shown the ability to rapidly detect the vibration state of C-H bonds in triglycerides. The aim of this study is to determine whether conventional Raman spectroscopy can reliably detect and quantify fat in an animal model of liver steatosis. Mice and rats fed a methionine and choline-deficient (MCD) and control diets were sacrificed on one, two, three and four weeks' time points. A confocal Raman microscope, a commercial Raman (iRaman) fiber optic probe and a highly sensitive Raman fiber optic probe system, the latter utilizing a 785 nm excitation laser, were used to detect changes in the Raman spectra of steatotic mouse livers. Thin layer chromatography was used to assess the triglyceride content of liver specimens, and sections were scored blindly for fat content using histological examination. Principal component analysis (PCA) of Raman spectra was used to extract the principal components responsible for spectroscopic differences with MCD week (time on MCD diet). Confocal Raman microscopy revealed the presence of saturated fats in mice liver sections. A commercially available handheld Raman spectroscopy probe could not distinguish the presence of fat in the liver whereas our specially designed, high throughput Raman system could clearly distinguish lobe-specific changes in fat content. In the left lobe in particular, the Raman PC scores exhibited a significant correlation (R(2) = 0.96) with the gold standard, blinded scoring by histological examination. The specially designed, high throughput Raman system can be used for clinical purposes. Its application to the field of transplantation would enable surgeons to determine the hepatic fat content of the donor's liver in the field prior to proceeding with organ retrieval. Next steps include validating these results in a prospective analysis of human liver transplantation implant biopsies.
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Hígado Graso/diagnóstico , Tecnología de Fibra Óptica , Espectrometría Raman/instrumentación , Animales , Modelos Animales de Enfermedad , Endoscopía , Hígado Graso/metabolismo , Hígado Graso/patología , Humanos , Masculino , Ratones , RatasRESUMEN
Stem cells offer tremendous opportunities for regenerative medicine. Over the past decade considerable research has taken place to identify and characterize the differentiation states of stem cells in culture. Raman micro-spectroscopy has emerged as an ideal technology since it is fast, nondestructive, and does not require potentially toxic dyes. Raman spectroscopy systems can also be incorporated into confocal microscope imaging systems allowing spectra to be obtained from below the tissue surface. Thus there is significant potential for monitoring stem cells in living tissue. Stem cells that reside in hair follicles are suitable for testing this possibility since they are close to the skin surface, and typically clustered around the bulge area. One of the first steps needed would be to obtain Raman micro-spectra from stem cells located in thin sections of tissue, and then see whether these spectra are clearly different from those of the surrounding differentiated cells. To facilitate this test, standard 5 µm thick sections of murine skin tissue were stained to identify the location of hair follicle stem cells and their progeny. Raman spectra were then obtained from adjacent cells in a subsequent unstained 10 µm thick section. The spectra revealed significant differences in peak intensities associated with nucleic acids, proteins, lipids and amino acids. Statistical analyses of the Raman micro-spectra identified stem cells with 98% sensitivity and 94% specificity, as compared with a CD34 immunostaining gold standard. Furthermore analyses of the spectral variance indicated differences in cellular dynamics between the two cell groups. This study shows that Raman micro-spectroscopy has a potential role in identifying adult follicle stem cells, laying the groundwork for future applications of hair follicle stem cells and other somatic stem cells in situ.
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Folículo Piloso/citología , Espectrometría Raman/métodos , Células Madre/citología , Animales , Femenino , Ratones , Ratones Endogámicos C3HRESUMEN
Raman systems have tremendous potential as adjunct devices for endoscopes to improve the identification of early colon cancers. However, the traditional low frequency (LF) measurement range has several obstacles that make it challenging to develop a routine clinical tool. An alternative is to use high frequency (HF) range. To test this idea Raman spectra were obtained in both the LF and HF ranges from the same colon lesions. Multivariate analyses predicted the pathology with high sensitivity and specificity for both the LF and HF data sets. This suggests that Raman systems that measure HF spectra, and are simpler to adopt into the clinic, could be used in vivo to improve the identification of neoplastic lesions.
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Algoritmos , Neoplasias del Colon/diagnóstico , Diagnóstico por Computador/métodos , Espectrometría Raman/métodos , Interpretación Estadística de Datos , Humanos , Reproducibilidad de los Resultados , Sensibilidad y EspecificidadRESUMEN
We previously developed a hollow-core photonic crystal fiber (HCPCF) based Raman scattering enhancement technique for gas/human breath analysis. It enhances photon-gas molecule interactions significantly but is still based on CW laser excitation spontaneous Raman scattering, which is a low-probability phenomenon. In this work, we explored nanosecond/sub-nanosecond pulsed laser excitation in HCPCF based fiber enhanced Raman spectroscopy (FERS) and successfully induced stimulated Raman scattering (SRS) enhancement. Raman measurements of simple and complex gases were performed using the new system to assess its feasibility for gas analysis. We studied the gas Raman scattering characteristics, the relationship between Raman intensities and pump energies, and the energy threshold for the transition from spontaneous Raman scattering to SRS. H2, CO2, and propene (C3H6) were used as test gases. Our results demonstrated that a single-beam pulsed pump combined with FERS provides an effective Raman enhancement technique for gas analysis. Furthermore, an energy threshold for SRS initiation was experimentally observed. The SRS-capable FERS system, utilizing a single-beam pulsed pump, shows great potential for analyzing complex gases such as propene, which is a volatile organic compound (VOC) gas, serving as a biomarker in human breath for lung cancer and other human diseases. This work contributes to the advancement of gas analysis and opens alternative avenues for exploring novel Raman enhancement techniques.
RESUMEN
Raman enhancement techniques are essential for gas analysis to increase the detection sensitivity of a Raman spectroscopy system. We have developed an efficient Raman enhancement technique called the collision-enhanced Raman scattering (CERS), where the active Raman gas as the analyte is mixed with a buffer gas inside the hollow-core photonic-crystal fiber (HCPCF) of a fiber-enhanced Raman spectroscopy (FERS) system. This results in an enhanced Raman signal from the analyte gas. In this study, we first showed that the intensity of the 587 cm-1 stimulated Raman scattering (SRS) peak of H2 confined in an HCPCF is enhanced by as much as five orders of magnitude by mixing with a buffer gas such as helium or N2. Secondly, we showed that the magnitudes of Raman enhancement depend on the type of buffer gas, with helium being more efficient compared to N2. This makes helium a favorable buffer gas for CERS. Thirdly, we applied CERS for Raman measurements of propene, a metabolically interesting volatile organic compound (VOC) with an association to lung cancer. CERS resulted in a substantial enhancement of propene Raman peaks. In conclusion, the CERS we developed is a simple and efficient Raman-enhancing mechanism for improving gas analysis. It has great potential for application in breath analysis for lung cancer detection.
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Neoplasias Pulmonares , Espectrometría Raman , Humanos , Espectrometría Raman/métodos , Helio , Óptica y FotónicaRESUMEN
The management of cancer in the periphery lung is in critical need of new strategies. Here, the development and test of a novel miniature Raman probe capable of navigating the peripheral lung architecture is reported. The probe was 1.35 mm in diameter, with a minimum bend radius of 13 mm and had a large light collection area for its size. Peripheral lung Raman spectra were successfully obtained from normal tissue and cancerous nodule using the probe coupled to a home-made rapid Raman spectroscopy system with a fast integration time of 1 second and a low excitation power of 15 mW. This is the first time in vivo Raman spectra from the periphery lung being reported. The collected spectra showed lipid, protein and deoxyhemoglobin signatures that might be useful for classifying pathology. Large scale clinical study is planned to confirm the utility of this new technology for improving periphery lung cancer detection. Left: Radial ultrasound image of a peripheral lung nodule: size given by crosshairs D1 and D2. Right: Truncated Raman spectra of a cancerous nodule, whole blood, and normal peripheral airway tissue. Spectra were shifted on intensity scale for clarity.
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Detección Precoz del Cáncer/instrumentación , Neoplasias Pulmonares/diagnóstico , Miniaturización/instrumentación , Espectrometría Raman/instrumentación , Diseño de Equipo , HumanosRESUMEN
Currently the most sensitive method for localizing lung cancers in central airways is autofluorescence bronchoscopy (AFB) in combination with white light bronchoscopy (WLB). The diagnostic accuracy of WLB + AFB for high grade dysplasia (HGD) and carcinoma in situ is variable depending on physician's experience. When WLB + AFB are operated at high diagnostic sensitivity, the associated diagnostic specificity is low. Raman spectroscopy probes molecular vibrations and gives highly specific, fingerprint-like spectral features and has high accuracy for tissue pathology classification. In this study we present the use of a real-time endoscopy Raman spectroscopy system to improve the specificity. A spectrum is acquired within 1 second and clinical data are obtained from 280 tissue sites (72 HGDs/malignant lesions, 208 benign lesions/normal sites) in 80 patients. Using multivariate analyses and waveband selection methods on the Raman spectra, we have demonstrated that HGD and malignant lung lesions can be detected with high sensitivity (90%) and good specificity (65%).
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Detección Precoz del Cáncer/métodos , Neoplasias Pulmonares/diagnóstico , Espectrometría Raman , Broncoscopía , Humanos , Pulmón , Sensibilidad y EspecificidadRESUMEN
Confocal micro-Raman spectroscopy is used to probe the nuclei of normal human epidermal cells and epidermally derived cancer cells from nodular basal cell carcinomas. Clear differences are seen between the spectra. The nuclei of tumor cells appear to have different contributions from nucleic acids, histones, and proteins with an actin-like spectrum than those of normal epidermal cells. Changes in the contribution of DNA to the spectra are consistent with the staining of conventional histopathologic specimens. We also obtain spectra of the dermis, where it is found that the dermis close to tumor boundaries is not simply deficient in collagen, but shows signs of structural changes as well.
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Carcinoma Basocelular/metabolismo , Carcinoma Basocelular/patología , Núcleo Celular/metabolismo , Núcleo Celular/patología , Colágeno/análisis , Microscopía Confocal/instrumentación , Espectrometría Raman/instrumentación , Diseño de Equipo , Análisis de Falla de Equipo , Humanos , Microscopía Confocal/métodos , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/patología , Espectrometría Raman/métodos , Células Tumorales CultivadasRESUMEN
In this study, we used IGH sequence analysis to assess the maturational status of Waldenstrom's (WM) macroglobulinemia and its putative precursor immunoglobulin (Ig)-M monoclonal gammopathy of undetermined significance (MGUS). IGH sequence analysis was performed using standard methods in 23 cases (20 WM and 3 IgM MGUS as defined by consensus panel criteria). Waldenstrom's macroglobulinemia cases were characterized by heavily mutated IGH genes (median, 6.3%; range, 3.8%-13.9%) but without intraclonal variation (ICV). IgM MGUS was similarly characterized by somatic hypermutation (median, 7.5%; range, 7%-7.7%), but ICV was evident in 1 of the 3 cases. We would therefore conclude that WM is characterized by somatic hypermutation without ICV, which supports a derivation from postgerminal center/memory B cells. IgM MGUS is also characterized by somatic hypermutation but, in a manner similar to IgA/IgG MGUS, can be associated with ICV, although the significance of this remains unclear.
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Cadenas Pesadas de Inmunoglobulina/química , Inmunoglobulina M/química , Mutación , Paraproteinemias/inmunología , Macroglobulinemia de Waldenström/inmunología , Anciano , Femenino , Reordenamiento Génico , Humanos , Cadenas Pesadas de Inmunoglobulina/genética , Inmunoglobulina M/genética , Masculino , Persona de Mediana Edad , Paraproteinemias/genética , Análisis de Secuencia de ADN , Macroglobulinemia de Waldenström/genéticaRESUMEN
The objective of this study was to build and test an adjunct system to a colonoscope for in vivo measurement of Raman spectra from colon tissue for potentially improving the detection of early cancers. The novelty of this system was that low cost fibre optic probes were used, without the addition of expensive optical filters. Good quality in vivo Raman spectra were successfully obtained with a 1 s integration time in the high frequency (HF) range from normal tissue and polyps of patients during a colonoscopy. The polyps were subsequently removed, and their pathology determined. The acquired in vivo Raman spectra showed clear changes between tissue with normal and tubular adenoma pathology. Further clinical study with this low cost HF Raman probe is warranted to fully test its clinical utility.
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Neoplasias del Colon/diagnóstico , Detección Precoz del Cáncer/métodos , Endoscopía , Espectrometría Raman/métodos , Detección Precoz del Cáncer/instrumentación , Humanos , Espectrometría Raman/instrumentaciónRESUMEN
CASE DESCRIPTION: A 7-year-old Quarter Horse gelding was hospitalized in Ocala, Fla, because of lethargy, fever, anorexia, and swelling of distal aspects of the limbs. A tentative diagnosis of equine piroplasmosis (EP) was made on the basis of examination of a blood smear. The case was reported to the Florida State Veterinarian, and infection with Babesia equi was confirmed. The subsequent investigation included quarantine and testing of potentially exposed horses for B equi and Babesia caballi infections, tick surveillance, and owner-agent interviews. CLINICAL FINDINGS: 210 horses on 25 premises were tested for infection with EP pathogens. Twenty B equi-infected horses on 7 premises were identified; no horses tested positive for B caballi. Seven horses, including the index case, had clinical findings consistent with EP Dermacentor variabilis was considered the only potential tick vector for B equi collected, and all D variabilis specimens tested negative for Babesia organisms via PCR assay. Results of the epidemiological investigation suggested that B equi was spread by use of shared needles and possibly blood transfusions. All horses that tested positive were involved in nonsanctioned Quarter Horse racing, and management practices were thought to pose substantial risk of transmission of blood-borne pathogens. TREATMENT AND OUTCOME: Final outcome of B equi-infected horses was euthanasia, death from undetermined causes, or shipment to a US federal research facility. CLINICAL RELEVANCE: This investigation highlights the importance of collaboration between private veterinary practitioners, state veterinary diagnostic laboratories, and regulatory officials in the recognition, containment, and eradication of foreign animal disease.
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Babesiosis/veterinaria , Brotes de Enfermedades/veterinaria , Enfermedades de los Caballos/epidemiología , Crianza de Animales Domésticos , Animales , Babesia/aislamiento & purificación , Babesiosis/epidemiología , Babesiosis/mortalidad , Babesiosis/transmisión , Femenino , Florida/epidemiología , Enfermedades de los Caballos/mortalidad , Enfermedades de los Caballos/parasitología , Enfermedades de los Caballos/transmisión , Caballos , MasculinoRESUMEN
INTRODUCTION: Preneoplastic lesions of the bronchial tree have a high probability of developing into malignant tumors. Currently, the best method for localizing them for further treatment is a combined white light bronchoscopy (WLB) and autofluorescence bronchoscopy (AFB) (WLB + AFB). The average specificity from large clinical trials for this combined detection method is approximately 60%, leading to many false positives. The object of this study is to determine whether adding point laser Raman spectroscopy (LRS) to a WLB + AFB has the potential to improve the specificity of preneoplastic lesion detection and what the implication is to the detection sensitivity. METHODS: An LRS system was developed to collect real-time, in vivo lung spectra with a fiber optic catheter passed down the instrument channel of a bronchoscope. WLB + AFB imaging modalities were used to identify lesions from 26 subjects, from which 129 Raman spectra were measured. Multivariate statistical analyses were performed on the spectra with a leave-one-out crossvalidation. RESULTS: Clear in vivo Raman spectra were obtained in 1 second. The location of individual Raman peaks in the spectra correlated well with the known positions of Raman peaks generated by lipids, proteins, and water molecules. Preneoplastic lesions were detected with a sensitivity of 96% and a specificity of 91%. CONCLUSION: Adding point LRS analysis to WLB + AFB imaging has the ability to detect preneoplastic lesions in real time with high sensitivity and specificity. The use of LRS has great potential for substantially reducing the number of false-positive biopsies associated with WLB + AFB with very little reduction in the detection sensitivity.
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Neoplasias de los Bronquios/diagnóstico , Broncoscopía , Carcinoma in Situ/diagnóstico , Carcinoma de Células Escamosas/diagnóstico , Rayos Láser , Lesiones Precancerosas/diagnóstico , Espectrometría Raman , Biopsia , Reacciones Falso Positivas , Fluorescencia , Humanos , Proyectos Piloto , Pronóstico , Factores de Riesgo , Sensibilidad y Especificidad , FumarRESUMEN
A near-infrared Raman system was developed to collect real-time in vivo human lung spectra. The excitation light and the emission were guided to and from the tissue surface by a reusable fiber catheter passed down the instrument channel of a bronchoscope. Two-stage filtering was used to reduce laser noise, fluorescence, and Raman emissions from the fibers. A second fiber bundle guided the emission to a spectrometer where the fibers, in a round packing geometry, were spread out to form a parabolic arc that improved the signal-to-noise ratio 20-fold, facilitating real-time spectral measurements. Preliminary clinical tests show that clear and reliable Raman spectra can be obtained.
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Endoscopía/métodos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Pulmón/patología , Espectrometría Raman/métodos , Biopsia , Broncoscopios , Broncoscopía/métodos , Calibración , Diseño de Equipo , Humanos , Rayos Láser , Sensibilidad y Especificidad , Espectrometría de Fluorescencia/métodosRESUMEN
We propose to combine field imaging endoscopy with point spectral analysis for improving the overall diagnostic accuracy in clinical lung cancer detection. For this purpose, we developed an integrated endoscopy system that uses autofluorescence imaging and white light reflectance imaging to obtain high diagnostic sensitivity, while at the same time uses non-contact point reflectance/fluorescence spectroscopy to reduce false positive biopsies, thus, achieve high diagnostic specificity. A pilot clinical test on 22 lung patients demonstrated that using this system the malignant lung lesions can be differentiated from the benign lesions with both diagnostic sensitivity and specificity of better than 80%. To further reduce the number of false positive diagnosis and allow even higher diagnostic accuracies, we have also developed an endoscopic laser Raman probe for in vivo real-time biochemical analysis of the suspicious tissue areas identified by the field imaging modalities (white light imaging and autofluorescence imaging). Preliminary Raman spectroscopy results will be reported at the conference.