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We have developed a noncontact, photothermal materials characterization method based on visible-light speckle imaging. This technique is applied to remotely measure the infrared absorption spectra of materials and to discriminate materials based on their thermal conductivities. A wavelength-tunable (7.5-8.7 µm), intensity-modulated, quantum cascade pump laser and a continuous-wave 532 nm probe laser illuminate a sample surface such that the two laser spots overlap. Surface absorption of the intensity-modulated pump laser induces a time-varying thermoelastic surface deformation, resulting in a time-varying 532 nm scattering speckle field from the surface. The speckle modulation amplitude, derived from a series of visible camera images, is found to correlate with the amplitude of the surface motion. By tuning the pump laser's wavelength over a molecular absorption feature, the amplitude spectrum of the speckle modulation is found to correlate to the IR absorption spectrum. As an example, we demonstrate this technique for spectroscopic identification of thin polymeric films. Furthermore, by adjusting the rate of modulation of the pump beam and measuring the associated modulation transfer to the visible speckle pattern, information about the thermal time constants of surface and sub-surface features can be revealed. Using this approach, we demonstrate the ability to distinguish between different materials (including metals, semiconductors, and insulators) based on differences in their thermal conductivities.
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Conventional histopathology with hematoxylin & eosin (H&E) has been the gold standard for histopathological diagnosis of a wide range of diseases. However, it is not performed in vivo and requires thin tissue sections obtained after tissue biopsy, which carries risk, particularly in the central nervous system. Here we describe the development of an alternative, multicolored way to visualize tissue in real-time through the use of coherent Raman imaging (CRI), without the use of dyes. CRI relies on intrinsic chemical contrast based on vibrational properties of molecules and intrinsic optical sectioning by nonlinear excitation. We demonstrate that multicolor images originating from CH(2) and CH(3) vibrations of lipids and protein, as well as two-photon absorption of hemoglobin, can be obtained with subcellular resolution from fresh tissue. These stain-free histopathological images show resolutions similar to those obtained by conventional techniques, but do not require tissue fixation, sectioning or staining of the tissue analyzed.
Assuntos
Rastreamento de Células/métodos , Técnicas de Preparação Histocitológica , Análise Espectral Raman/métodos , Tomografia de Coerência Óptica/métodos , Animais , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Doenças Desmielinizantes/patologia , Modelos Animais de Doenças , Hemoglobinas/química , Humanos , Lipídeos/química , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Nus , Proteínas/química , Coloração e Rotulagem , Acidente Vascular Cerebral/patologia , Tomografia de Coerência Óptica/instrumentaçãoRESUMO
We present an approach for rational design and optimization of plasmonic arrays for ultrasensitive surface enhanced infrared absorption (SEIRA) spectroscopy of specific protein analytes. Motivated by our previous work that demonstrated sub-attomole detection of surface-bound silk fibroin [Proc. Natl. Acad. Sci. U.S.A. 106, 19227 (2009)], we introduce here a general framework that allows for the numerical optimization of metamaterial sensor designs in order to maximize the absorbance signal. A critical feature of our method is the explicit compensation for the perturbative effects of the analyte's refractive index which alters the resonance frequency and line-shape of the metamaterial response, thereby leading to spectral distortion in SEIRA signatures. As an example, we leverage our method to optimize the geometry of periodic arrays of plasmonic nanoparticles on both Si and CaF2 substrates. The optimal geometries result in a three-order of magnitude absorbance enhancement compared to an unstructured Au layer, with the CaF2 substrate offering an additional factor of three enhancement in absorbance over a traditional Si substrate. The latter improvement arises from increase of near-field intensity over the Au nanobar surface for the lower index substrate. Finally, we perform sensitivity analysis for our optimized arrays to predict the effects of fabrication imperfections. We find that <20% deviation from the optimized absorbance response is readily achievable over large areas with modern nanofabrication techniques.
Assuntos
Desenho Assistido por Computador , Modelos Teóricos , Nanotecnologia/instrumentação , Refratometria/instrumentação , Espectrofotometria Infravermelho/instrumentação , Ressonância de Plasmônio de Superfície/instrumentação , Simulação por Computador , Desenho de Equipamento , Análise de Falha de Equipamento , Luz , Espalhamento de RadiaçãoRESUMO
Coherent Raman scattering methods allow for label-free imaging of tissue with chemical contrast and high spatial and temporal resolution. However, their imaging depth in scattering tissue is limited to less than 1 mm, requiring the development of endoscopes to obtain images deep inside the body. Here, we describe a coherent Raman endoscope that provides stimulated Raman scattering images at seven frames per second using a miniaturized fiber scanner, a custom-designed objective lens, and an optimized scheme for collection of scattered light from the tissue. We characterize the system and demonstrate chemical selectivity in mouse tissue images.
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Endoscopia/métodos , Análise Espectral Raman/métodos , Animais , Camundongos , PeleRESUMO
Efficient drug delivery to the skin is essential for the treatment of major dermatologic diseases, such as eczema, psoriasis and acne. However, many compounds penetrate the skin barrier poorly and require optimized formulations to ensure their bioavailability. Here, stimulated Raman scattering (SRS) microscopy, a recently developed, label-free chemical imaging tool, is used to acquire high resolution images of multiple chemical components of a topical formulation as it penetrates into mammalian skin. This technique uniquely provides label-free, nondestructive, three-dimensional images with high spatiotemporal resolution. It reveals novel features of (trans)dermal drug delivery in the tissue environment: different rates of drug penetration via hair follicles as compared to the intercellular pathway across the stratum corneum are directly observed, and the precipitation of drug crystals on the skin surface is visualized after the percutaneous penetration of the cosolvent excipient in the formulation. The high speed three-dimensional imaging capability of SRS thus reveals features that cannot be seen with other techniques, providing both kinetic information and mechanistic insight into the (trans)dermal drug delivery process.
Assuntos
Absorção Cutânea/fisiologia , Pele/metabolismo , Análise Espectral Raman/métodos , Administração Cutânea , Animais , Camundongos , Camundongos Endogâmicos BALB CRESUMO
We use the time-lens concept to demonstrate a new scheme for synchronization of two pulsed light sources for biological imaging. An all fiber, 1064 nm time-lens source is synchronized to a picosecond solid-state Ti: Sapphire mode-locked laser by using the mode-locked laser pulses as the clock. We demonstrate the application of this synchronized source for CARS and SRS imaging by imaging mouse tissues. Synchronized two wavelength pulsed source is an important technical difficulty for CARS and SRS imaging. The time-lens source demonstrated here may provide an all fiber, user friendly alternative for future SRS imaging.
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We present a novel intracavity frequency modulation scheme in a tunable, picosecond optical parametric oscillator (OPO). The OPO signal wavelength can be modulated with a depth of more than 10 nm at a rate of 38 MHz (one half its repetition rate). We discuss the design and construction of the light source and its application to the recently-developed frequency modulation coherent anti-Stokes Raman scattering (FM-CARS) and stimulated Raman scattering (SRS) techniques. The new light source allows for real time subtraction of the interfering background signal in coherent Raman imaging, yielding images with purely chemical contrast.
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Microscopia/métodos , Oscilometria/métodos , Análise Espectral Raman/métodos , Animais , Desenho de Equipamento , Cabelo , Lasers , Luz , Camundongos , Minoxidil/farmacologia , Óptica e Fotônica , Física/métodos , Espalhamento de Radiação , Pele/patologia , Fatores de TempoRESUMO
Liquid crystals are a class of industrially important materials whose optical properties make them useful particularly in display technology. Optical imaging of these materials provides information about their structure and physical properties. Coherent anti-Stokes Raman scattering (CARS) microscopy is used to provide three-dimensional chemical maps of liquid crystalline samples without the use of external labels. CARS is an optical imaging technique that derives contrast from Raman-active molecular vibrations in the sample. Compared to many other three-dimensional imaging techniques, CARS offers more rapid chemical characterization without the use of external dyes or contrast agents. The use of CARS to image chemical and orientational order in liquid crystals is demonstrated using several examples, and the limitations and benefits are discussed.
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Imaging in neuroscience has been dramatically impacted by the advent of multiphoton microscopy. Multiphoton-excited fluorescence (MPF) in combination with endogenous fluorophores or labeling by fluorescent molecules has proven to be particularly powerful. However, endogenous fluorescence is limited to relatively few molecular species, and practical labeling schemes do not exist for many classes of molecules. Coherent Raman scattering (CRS) techniques, including coherent anti-Stokes Raman scattering and stimulated Raman scattering, allow imaging without the need for staining or fluorescent labeling. Such label-free imaging is desirable in biomedical research, because labeling often perturbs the function of small metabolite and drug molecules and may be too toxic to use in vivo. CRS techniques have similar imaging parameters to MPF, making use of pulsed near-infrared lasers to deliver high-sensitivity, high spatial resolution in three dimensions and rapid image acquisition. In this introduction, we will discuss the basic principles of CRS imaging, present the instrumentation requirements for high-speed CRS imaging, and show an example of imaging brain tumors and healthy tissue based on their intrinsic vibrational signatures. This discussion is intended to introduce the benefits and tradeoffs associated with different CRS techniques and show one example of the powerful capabilities of label-free chemical imaging.
Assuntos
Química Encefálica , Encéfalo/fisiologia , Imagem Óptica/métodos , Análise Espectral Raman/métodos , Encéfalo/patologia , Imageamento Tridimensional/métodos , Imagem Óptica/instrumentação , Análise Espectral Raman/instrumentaçãoRESUMO
Label-free microscopy based on Raman scattering has been increasingly used in biomedical research to image samples that cannot be labeled or stained. Stimulated Raman scattering (SRS) microscopy allows signal amplification of the weak Raman signal for fast imaging speeds without introducing the nonresonant background and coherent image artifacts that are present in coherent anti-Stokes Raman scattering (CARS) microscopy. Here we present the Raman-induced Kerr effect (RIKE) as a contrast for label-free microscopy. RIKE allows us to measure different elements of the nonlinear susceptibility tensor, both the real and imaginary parts, by optical heterodyne detection (OHD-RIKE). OHD-RIKE microscopy provides information similar to polarization CARS (P-CARS) and interferometric CARS (I-CARS) microscopy, with a simple modification of the two-beam SRS microscopy setup. We show that, while OHD-RIKE microspectroscopy can be in principle more sensitive than SRS, it does not supersede SRS microscopy of heterogeneous biological samples, such as mouse skin tissue, because it is complicated by variations of linear birefringence across the sample.
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Microscopia/instrumentação , Animais , Epiderme/anatomia & histologia , Camundongos , Microscopia/métodos , Análise Espectral RamanRESUMO
The development of methods that allow microscale studies of complex biomaterials based on their molecular composition is of great interest to a wide range of research fields. We show that stimulated Raman scattering (SRS) microscopy is an excellent analytical tool to study distributions of different biomolecules in multiphasic systems. SRS combines the label-free molecular specificity of vibrational spectroscopy with an enhanced sensitivity due to coherent excitation of molecular vibrations. Compared to previous imaging studies using coherent anti-Stokes Raman scattering microscopy, the main advantage of SRS microscopy is the absence of the unwanted nonresonant background, which translates into a superior sensitivity and undistorted vibrational spectra. We compare spectra of complex materials obtained with stimulated Raman scattering and spontaneous Raman scattering in the crowded fingerprint region.We find that, as expected, there is excellent correspondence and that the SRS spectra are free from interference from background fluorescence. In addition, we show high-resolution imaging of the distributions of selected biomolecules, such as lipids and proteins, in food products with SRS microscopy.
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Biopolímeros/análise , Análise de Alimentos/instrumentação , Microscopia/instrumentação , Análise Espectral Raman/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Coloração e RotulagemRESUMO
Optical imaging in vivo with molecular specificity is important in biomedicine because of its high spatial resolution and sensitivity compared with magnetic resonance imaging. Stimulated Raman scattering (SRS) microscopy allows highly sensitive optical imaging based on vibrational spectroscopy without adding toxic or perturbative labels. However, SRS imaging in living animals and humans has not been feasible because light cannot be collected through thick tissues, and motion-blur arises from slow imaging based on backscattered light. In this work, we enable in vivo SRS imaging by substantially enhancing the collection of the backscattered signal and increasing the imaging speed by three orders of magnitude to video rate. This approach allows label-free in vivo imaging of water, lipid, and protein in skin and mapping of penetration pathways of topically applied drugs in mice and humans.
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Imagem Molecular/métodos , Pele/química , Pele/metabolismo , Análise Espectral Raman/métodos , Administração Cutânea , Animais , Capilares , Dimetil Sulfóxido/administração & dosagem , Dimetil Sulfóxido/farmacocinética , Epiderme/química , Epiderme/metabolismo , Eritrócitos/fisiologia , Humanos , Imageamento Tridimensional , Luz , Lipídeos , Masculino , Camundongos , Camundongos Nus , Pele/irrigação sanguínea , Fatores de Tempo , Vitamina A/administração & dosagem , Vitamina A/farmacocinética , ÁguaRESUMO
The development of methods that allow microscale studies of complex biomaterials based on their molecular composition is of great interest to a wide range of research fields. We show that stimulated Raman scattering (SRS) microscopy is an excellent analytical tool to study distributions of different biomolecules in multiphasic systems. SRS combines the label-free molecular specificity of vibrational spectroscopy with an enhanced sensitivity due to coherent excitation of molecular vibrations. Compared to previous imaging studies using coherent anti-Stokes Raman scattering microscopy, the main advantage of SRS microscopy is the absence of the unwanted nonresonant background, which translates into a superior sensitivity and undistorted vibrational spectra. We compare spectra of complex materials obtained with stimulated Raman scattering and spontaneous Raman scattering in the crowded fingerprint region. We find that, as expected, there is excellent correspondence and that the SRS spectra are free from interference from background fluorescence. In addition, we show high-resolution imaging of the distributions of selected biomolecules, such as lipids and proteins, in food products with SRS microscopy.
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Biopolímeros/análise , Análise de Alimentos/instrumentação , Microscopia/instrumentação , Análise Espectral Raman/instrumentação , Desenho de Equipamento , Análise de Falha de Equipamento , Coloração e RotulagemRESUMO
We report a high-power picosecond fiber pump laser system for coherent Raman microscopy (CRM). The fiber laser system generates 3.5 ps pulses with 6 W average power at 1030 nm. Frequency doubling yields more than 2 W of green light, which can be used to pump an optical parametric oscillator to produce the pump and the Stokes beams for CRM. Detailed performance data on the laser and the various wavelength conversion steps are discussed, together with representative CRM images of fresh animal tissue obtained with the new source.
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Microscopia/instrumentação , Microscopia/métodos , Animais , Lasers , Camundongos , Pele/citologia , Pele/metabolismo , Análise Espectral Raman , Temperatura , Fatores de TempoRESUMO
Label-free chemical contrast is highly desirable in biomedical imaging. Spontaneous Raman microscopy provides specific vibrational signatures of chemical bonds, but is often hindered by low sensitivity. Here we report a three-dimensional multiphoton vibrational imaging technique based on stimulated Raman scattering (SRS). The sensitivity of SRS imaging is significantly greater than that of spontaneous Raman microscopy, which is achieved by implementing high-frequency (megahertz) phase-sensitive detection. SRS microscopy has a major advantage over previous coherent Raman techniques in that it offers background-free and readily interpretable chemical contrast. We show a variety of biomedical applications, such as differentiating distributions of omega-3 fatty acids and saturated lipids in living cells, imaging of brain and skin tissues based on intrinsic lipid contrast, and monitoring drug delivery through the epidermis.
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Imageamento Tridimensional/métodos , Lipídeos/análise , Microscopia/métodos , Análise Espectral Raman , Animais , Linhagem Celular Tumoral , Corpo Caloso/química , Corpo Caloso/citologia , Dimetil Sulfóxido/administração & dosagem , Dimetil Sulfóxido/farmacocinética , Ácido Eicosapentaenoico/metabolismo , Epiderme/química , Epiderme/metabolismo , Epiderme/ultraestrutura , Humanos , Camundongos , Neurônios/ultraestrutura , Sensibilidade e Especificidade , Pele/química , Pele/ultraestrutura , Tretinoína/administração & dosagem , Tretinoína/farmacocinética , Vitamina A/análise , Vitamina A/químicaRESUMO
We demonstrate a new approach to coherent anti-Stokes Raman scattering (CARS) microscopy that significantly increases the detection sensitivity. CARS signals are generated by collinearly overlapped, tightly focused, and raster scanned pump and Stokes laser beams, whose difference frequency is rapidly modulated. The resulting amplitude modulation of the CARS signal is detected through a lock-in amplifier. This scheme efficiently suppresses the nonresonant background and allows for the detection of far fewer vibrational oscillators than possible through existing CARS microscopy methods.
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Aumento da Imagem/métodos , Interpretação de Imagem Assistida por Computador/métodos , Lasers , Microscopia/métodos , Análise Espectral Raman/métodos , Tomografia de Coerência Óptica/métodos , Vibração , Luz , Imagens de Fantasmas , Reprodutibilidade dos Testes , Espalhamento de Radiação , Sensibilidade e EspecificidadeRESUMO
We have recorded the vibrational absorption spectrum of 1,1,1,2-tetrafluoroethane (HFC-134a) in the fundamental and first five CH-stretching overtone regions with the use of Fourier transform infrared, dispersive long-path, intracavity laser photoacoustic, and cavity ringdown spectroscopies. We compare our measured total oscillator strengths in each region with intensities calculated using an anharmonic oscillator local mode model. We calculate intensities with 1D, 2D, and 3D Hamiltonians, including one or two CH stretches and two CH stretches with the HCH bending mode, respectively. The dipole moment function is calculated ab initio with self-consistent-field Hartree-Fock and density functional theories combined with double- and triple-zeta-quality basis sets. We find that the basis set choice affects the total intensity more than the choice of the Hamiltonian. We achieve agreement between the calculated and measured total intensities of approximately a factor of 2 or better for the fundamental and first five overtones.