RESUMEN
Due to its high molecular specificity, Raman spectroscopy is a well-established analytical tool. Usually the inelastically scattered Raman light is spectrally dispersed by a spectrometer. Here, we present an alternative method, using an optical fiber as dispersive element. As the group velocity within the fiber is wavelength-dependent, different Raman bands arrive at different times at the detector. In combination with time-correlated single-photon counting, Raman spectra can be measured in the time domain. As detector we implemented a Superconducting Nanowire Single-Photon Detector (SNSPD), which possesses a timing accuracy of about 20 ps. Within this contribution we show first results of Raman spectra measured in the time domain using gradient index fibers of varying length.
RESUMEN
In this contribution we present a dual modality fiber optic probe combining fluorescence lifetime imaging (FLIm) and Raman spectroscopy for in vivo endoscopic applications. The presented multi-spectroscopy probe enables efficient excitation and collection of fluorescence lifetime signals for FLIm in the UV/visible wavelength region, as well as of Raman spectra in the near-IR for simultaneous Raman/FLIm imaging. The probe was characterized in terms of its lateral resolution and distance dependency of the Raman and FLIm signals. In addition, the feasibility of the probe for in vivo FLIm and Raman spectral characterization of tissue was demonstrated. Graphical Abstract An image comparison between FLIm and Raman spectroscopy acquired with the bimodal probe onseveral tissue samples.
Asunto(s)
Tecnología de Fibra Óptica/instrumentación , Imagen Óptica/instrumentación , Espectrometría Raman/instrumentación , Animales , Química Encefálica , Endoscopía/instrumentación , Diseño de Equipo , Humanos , Ratas Endogámicas F344RESUMEN
Three important technical innovations are reported here towards Raman-activated cell sorting. Firstly, a microfluidic chip made of quartz is introduced which integrates injection of single cells, trapping by laser fibres and sorting of cells. Secondly, a chip holder was designed to provide simple, accurate and stable adjustment of chips, microfluidic connections and the trapping laser fibres. The new setup enables to the collection of Raman spectra of single cells at 785 nm excitation with 10 s exposure time. Lastly, a new type of modelling the various background contributions is described, improving Raman-based cell identification by the classification algorithm linear discriminant analysis. Mean sensitivity and specificity determined by iterated 10-fold cross validation were 96 and 99 %, respectively, for the distinction of leucocytes extracted from blood, breast cancer cells BT-20 and MCF-7, and leukaemia cells OCI-AML3.
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Células/química , Microfluídica/métodos , Espectrometría Raman/métodos , Línea Celular Tumoral , Humanos , Microfluídica/instrumentación , Pinzas Ópticas , Cuarzo , Espectrometría Raman/instrumentaciónRESUMEN
Visualization as well as characterization of inner arterial plaque depositions is of vital diagnostic interest, especially for the early recognition of vulnerable plaques. Established clinical techniques provide valuable visual information but cannot deliver information about the chemical composition of individual plaques. Here, we employ Raman-probe spectroscopy to characterize the plaque compositions of arterial walls on a rabbit model in vivo, using a miniaturized filtered probe with one excitation and 12 collection fibers integrated in a 1 mm sleeve. Rabbits were treated with a cholesterol-enriched diet. The methodology can improve the efficiency of animal experiments and shows great potential for applications in cardiovascular research. In order to further characterize the plaque depositions visually, coherent anti-Stokes Raman scattering (CARS) microscopy images have been acquired and are compared with the Raman-probe results.
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Placa Aterosclerótica/química , Espectrometría Raman , Animales , Aorta/patología , Dieta Alta en Grasa , Modelos Animales de Enfermedad , Masculino , Microscopía , Miniaturización , Placa Aterosclerótica/patología , ConejosRESUMEN
In the last years a variety of fiber optic Raman probes emerged, which are only partly suited for in vivo applications. The in vivo capability is often limited by the bulkiness of the probes. The size is associated with the required filtering of the probes, which is necessary due to Raman scattering inside the fibers. We employed in-line fiber Bragg gratings (FBG) as notch filter for the collection path and integrated them in a novel type of Raman probe. Multicore singlemode fibers (MCSMF) were designed and drawn integrating 19 singlemode cores to achieve better collection efficiency. A Raman probe was assembled with one excitation fiber and six MCSMF with inscribed FBGs as collection fibers. The probe was characterized regarding Raman background suppression, collection efficiency, and distance dependence. First Raman measurements on brain tissue are presented.
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Química Encefálica , Tecnología de Fibra Óptica/instrumentación , Imagen Molecular/instrumentación , Refractometría/instrumentación , Espectrometría Raman/instrumentación , Transductores , Animales , Diseño de Equipo , Análisis de Falla de Equipo , Porcinos , Integración de SistemasRESUMEN
Intravascular imaging techniques provide detailed specification about plaque appearance and morphology, but cannot deliver information about the biochemical composition of atherosclerotic plaques. As the biochemical composition is related to the plaque type, important aspects such as the risk of a plaque rupture and treatment are still difficult to assess. Currently, various spectroscopic techniques are tested for potential applications for the chemical analysis of plaque depositions. Here, we employ Raman spectroscopy in combination with optical coherence tomography (OCT) for the characterization of plaques on rabbits in vivo. Experiments were carried out on New Zealand white rabbits treated with a fat- and cholesterol-enriched diet, using a Raman probe setup with a 785-nm multimode laser as an excitation source. Subsequently, OCT images were acquired with a swept source at 1305±55 nm at 22.6 mW. Raman spectra were recorded from normal regions and regions with early plaque formations. The probe positioning was monitored by x-ray angiography. The spectral information identified plaque depositions consisting of lipids, with triglycerides as the major component. Afterward, OCT images of the spectroscopically investigated areas were obtained. The spectral information correlates well with the observed intravascular morphology and is in good agreement with histology. Raman spectroscopy can provide detailed biochemical specification of atherosclerotic plaques.
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Placa Aterosclerótica/diagnóstico por imagen , Espectrometría Raman/métodos , Tomografía de Coherencia Óptica/métodos , Animales , Arteria Carótida Común/diagnóstico por imagen , Procedimientos Endovasculares , Diseño de Equipo , Masculino , Conejos , Espectrometría Raman/instrumentaciónRESUMEN
Over the past years it had been demonstrated that multimodal imaging combining the nonlinear modalities coherent anti-Stokes Raman scattering (CARS), two-photon excited auto-fluorescence (TPEF) and second harmonic generation (SHG) show a great potential for tissue diagnosis and tumor identification. To extend the applicability of this multimodal imaging approach for in-vivo tissue screening of difficult to access body regions the development of suitable fiber optic probes is required. Here we report about a novel CARS imaging fiber probe consisting of 10,000 coherent light guiding elements preserving the spatial relationship between the entrance and the output of the fiber. Therefore the scanning procedure can be shifted from the distal to the proximal end of the fiber probe and no moving parts or driving current are required to realize in-vivo CARS endoscopy.
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Dinámicas no Lineales , Fibras Ópticas , Imagen Óptica/instrumentación , Animales , Aorta/citología , Conejos , Espectrometría RamanRESUMEN
Fluorescence lifetime imaging (FLIm) and Raman spectroscopy are two promising methods to support morphological intravascular imaging techniques with chemical contrast. Both approaches are complementary and may also be used in combination with OCT/IVUS to add chemical specificity to these morphologic intravascular imaging modalities. In this contribution, both modalities were simultaneously acquired from two human coronary specimens using a bimodal probe. A previously trained SVM model was used to interpret the fluorescence lifetime data; integrated band intensities displayed in RGB false color images were used to interpret the Raman data. Both modalities demonstrate unique strengths and weaknesses and these will be discussed in comparison to histologic analyses from the two coronary arteries imaged.
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Aterosclerosis/diagnóstico por imagen , Vasos Coronarios/diagnóstico por imagen , Espectrometría de Fluorescencia , Espectrometría Raman , Humanos , Miocardio/patología , Imagen ÓpticaRESUMEN
Although carbon monoxide (CO) delivery materials (CORMAs) have been generated, remote-controlled delivery with light-activated CORMAs at a local site has not been achieved. In this work, a fiber optic-based CO delivery system is described in which the photoactive and water insoluble CO releasing molecule (CORM) manganese(i) tricarbonyl [(OC)3Mn(µ3-SR)]4 (R = nPr, 1) has been non-covalently embedded into poly(l-lactide-co-d/l-lactide) and poly(methyl methacrylate) non-woven fabrics via the electrospinning technique. SEM images of the hybrid materials show a porous fiber morphology for both polymer supports. The polylactide non-woven fabric was attached to a fiber optical device. In combination with a laser irradiation source, remote-controlled and light-triggered CO release at 405 nm excitation wavelength was achieved. The device enabled a high flexibility of the spatially and timely defined application of CO with the biocompatible hybrid fabric in aqueous media. The rates of liberated CO were adjusted with the light intensity of the laser. CO release was confirmed via ATR-IR spectroscopy, a portable electrochemical CO sensor and a heterogeneous myoglobin assay.
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An all-fibre based Raman-on-chip setup is introduced which enables analysis of solutions and trapped particles without microscopes or objectives. Beside the novel quartz microfluidic chip, innovative multi-core single-mode fibres with integrated fibre Bragg gratings are used for detection. The limit of quantitation is 7.5 mM for urea and 2.5 mM for nicotine with linear Raman spectroscopy. This is an improvement of more than two orders of magnitude compared with previous fibre-based microfluidic Raman detection schemes. Furthermore, our device was combined with optical traps to collect Raman-on-chip spectra of spherical polymer beads.
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Técnicas Analíticas Microfluídicas/métodos , Nicotina/análisis , Espectrometría Raman , Urea/análisis , Calibración , Técnicas Analíticas Microfluídicas/instrumentación , Técnicas Analíticas Microfluídicas/normas , Nicotina/normas , Soluciones/química , Urea/normasRESUMEN
Raman spectroscopy has been recognized to be a powerful tool for label-free discrimination of cells. Sampling methods are under development to utilize the unique capabilities to identify cells in body fluids such as saliva, urine or blood. The current study applied optical traps in combination with Raman spectroscopy to acquire spectra of single cells in microfluidic glass channels. Optical traps were realized by two 1070 nm single mode fibre lasers. Microflows were controlled by a syringe pump system. A novel microfluidic glass chip was designed to inject single cells, modify the flow speed, accommodate the laser fibres and sort cells after Raman based identification. Whereas the integrated microchip setup used 514 nm for excitation of Raman spectra, a quartz capillary setup excited spectra with 785 nm laser wavelength. Classification models were trained using linear discriminant analysis to differentiate erythrocytes, leukocytes, acute myeloid leukaemia cells (OCI-AML3), and breast tumour cells BT-20 and MCF-7 with accuracies that are comparable with previous Raman experiments of dried cells and fixed cells in a Petri dish. Implementation into microfluidic environments enables a high degree of automation that is required to improve the throughput of the approach for Raman activated cell sorting.