RESUMEN
Label-free DNA imaging is highly desirable in biology and medicine to perform live imaging without affecting cell function and to obtain instant histological tissue examination during surgical procedures. Here we show a label-free DNA imaging method with stimulated Raman scattering (SRS) microscopy for visualization of the cell nuclei in live animals and intact fresh human tissues with subcellular resolution. Relying on the distinct Raman spectral features of the carbon-hydrogen bonds in DNA, the distribution of DNA is retrieved from the strong background of proteins and lipids by linear decomposition of SRS images at three optimally selected Raman shifts. Based on changes on DNA condensation in the nucleus, we were able to capture chromosome dynamics during cell division both in vitro and in vivo. We tracked mouse skin cell proliferation, induced by drug treatment, through in vivo counting of the mitotic rate. Furthermore, we demonstrated a label-free histology method for human skin cancer diagnosis that provides comparable results to other conventional tissue staining methods such as H&E. Our approach exhibits higher sensitivity than SRS imaging of DNA in the fingerprint spectral region. Compared with spontaneous Raman imaging of DNA, our approach is three orders of magnitude faster, allowing both chromatin dynamic studies and label-free optical histology in real time.
Asunto(s)
ADN/análisis , Microscopía , Neoplasias Cutáneas/diagnóstico , Espectrometría Raman , Animales , División Celular , Núcleo Celular/metabolismo , Proliferación Celular , ADN/química , Diagnóstico por Imagen , Femenino , Células HeLa , Humanos , Procesamiento de Imagen Asistido por Computador , Lípidos/química , Ratones , Ratones Desnudos , Mitosis , Neoplasias Cutáneas/metabolismoRESUMEN
Stimulated Raman Scattering microscopy allows label-free chemical imaging and has enabled exciting applications in biology, material science, and medicine. It provides a major advantage in imaging speed over spontaneous Raman scattering and has improved image contrast and spectral fidelity compared to coherent anti-Stokes Raman. Wider adoption of the technique has, however, been hindered by the need for a costly and environmentally sensitive tunable ultra-fast dual-wavelength source. We present the development of an optimized all-fibre laser system based on the optical synchronization of two picosecond power amplifiers. To circumvent the high-frequency laser noise intrinsic to amplified fibre lasers, we have further developed a high-speed noise cancellation system based on voltage-subtraction autobalanced detection. We demonstrate uncompromised imaging performance of our fibre-laser based stimulated Raman scattering microscope with shot-noise limited sensitivity and an imaging speed up to 1 frame/s.
RESUMEN
Stimulated Raman scattering (SRS) microscopy allows label-free chemical imaging based on vibrational spectroscopy. Narrowband excitation with picosecond lasers creates the highest signal levels and enables imaging speeds up to video-rate, but it sacrifices chemical specificity in samples with overlapping bands compared to broadband (multiplex) excitation. We develop a rapidly tunable picosecond optical parametric oscillator with an electro-optical tunable Lyot filter, and demonstrate multicolor SRS microscopy with synchronized line-by-line wavelength tuning to avoid spectral artifacts due to sample movement. We show sensitive imaging of three different kinds of polymer beads and live HeLa cells with moving intracellular lipid droplets.
Asunto(s)
Microscopía/métodos , Oscilometría/métodos , Espectrometría Raman/métodos , Artefactos , Diagnóstico por Imagen/métodos , Diseño de Equipo , Células HeLa , Humanos , Lípidos/química , Polimetil Metacrilato/química , Poliestirenos/química , Procesamiento de Señales Asistido por Computador , Triazinas/químicaRESUMEN
We present a fiber-format picosecond light source for coherent anti-Stokes Raman scattering microscopy. Pulses from a Yb-doped fiber amplifier are frequency converted by four-wave mixing (FWM) in normal-dispersion photonic crystal fiber to produce a synchronized two-color picosecond pulse train. We show that seeding the FWM process overcomes the deleterious effects of group-velocity mismatch and allows efficient conversion into narrow frequency bands. The source generates more than 160 mW of nearly transform-limited pulses tunable from 775 to 815 nm. High-quality coherent Raman images of animal tissues and cells acquired with this source are presented.
Asunto(s)
Microscopía/instrumentación , Fibras Ópticas , Espectrometría Raman/instrumentación , Animales , Encéfalo/citología , Células Epidérmicas , Fibroblastos/citología , Ratones , Ratas , Glándulas Sebáceas/citologíaRESUMEN
Imaging of nucleic acids is important for studying cellular processes such as cell division and apoptosis. A noninvasive label-free technique is attractive. Raman spectroscopy provides rich chemical information based on specific vibrational peaks. However, the signal from spontaneous Raman scattering is weak and long integration times are required, which drastically limits the imaging speed when used for microscopy. Coherent Raman scattering techniques, comprising coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy, overcome this problem by enhancing the signal level by up to five orders of magnitude. CARS microscopy suffers from a nonresonant background signal, which distorts Raman spectra and limits sensitivity. This makes CARS imaging of weak transitions in spectrally congested regions challenging. This is especially the case in the fingerprint region, where nucleic acids show characteristic peaks. The recently developed SRS microscopy is free from these limitations; excitation spectra are identical to those of spontaneous Raman and sensitivity is close to shot-noise limited. Herein we demonstrate the use of SRS imaging in the fingerprint region to map the distribution of nucleic acids in addition to proteins and lipids in single salivary gland cells of Drosophila larvae, and in single mammalian cells. This allows the imaging of DNA condensation associated with cell division and opens up possibilities of imaging such processes in vivo.
Asunto(s)
ADN/química , Espectrometría Raman , Animales , Línea Celular Tumoral , Drosophila melanogaster , Células HEK293 , Humanos , Glándulas Salivales/citologíaRESUMEN
Label-free microscopy with chemical contrast and high acquisition speed up to video-rate has recently been made possible by stimulated Raman scattering (SRS) microscopy. While SRS imaging offers superb sensitivity, the spectral specificity of the original narrowband implementation is limited, making distinguishing chemical species with overlapping Raman bands difficult. Here we present a highly specific imaging method that allows mapping of a particular chemical species in the presence of interfering species based on tailored multiplex excitation of its vibrational spectrum. This is done by spectral modulation of a broadband pump beam at a high-frequency (>1MHz), allowing detection of the stimulated Raman gain signal of the narrowband Stokes beam with high sensitivity. Using the scheme, we demonstrate quantification of cholesterol in the presence of lipids, and real-time three-dimensional spectral imaging of protein, stearic acid and oleic acid in live C.elegans.
RESUMEN
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.
Asunto(s)
Imagen Molecular/métodos , Piel/química , Piel/metabolismo , Espectrometría Raman/métodos , Administración Cutánea , Animales , Capilares , Dimetilsulfóxido/administración & dosificación , Dimetilsulfóxido/farmacocinética , Epidermis/química , Epidermis/metabolismo , Eritrocitos/fisiología , Humanos , Imagenología Tridimensional , Luz , Lípidos , Masculino , Ratones , Ratones Desnudos , Piel/irrigación sanguínea , Factores de Tiempo , Vitamina A/administración & dosificación , Vitamina A/farmacocinética , AguaRESUMEN
Fluorescence, that is, spontaneous emission, is generally more sensitive than absorption measurement, and is widely used in optical imaging. However, many chromophores, such as haemoglobin and cytochromes, absorb but have undetectable fluorescence because the spontaneous emission is dominated by their fast non-radiative decay. Yet the detection of their absorption is difficult under a microscope. Here we use stimulated emission, which competes effectively with the nonradiative decay, to make the chromophores detectable, and report a new contrast mechanism for optical microscopy. In a pump-probe experiment, on photoexcitation by a pump pulse, the sample is stimulated down to the ground state by a time-delayed probe pulse, the intensity of which is concurrently increased. We extract the miniscule intensity increase with shot-noise-limited sensitivity by using a lock-in amplifier and intensity modulation of the pump beam at a high megahertz frequency. The signal is generated only at the laser foci owing to the nonlinear dependence on the input intensities, providing intrinsic three-dimensional optical sectioning capability. In contrast, conventional one-beam absorption measurement exhibits low sensitivity, lack of three-dimensional sectioning capability, and complication by linear scattering of heterogeneous samples. We demonstrate a variety of applications of stimulated emission microscopy, such as visualizing chromoproteins, non-fluorescent variants of the green fluorescent protein, monitoring lacZ gene expression with a chromogenic reporter, mapping transdermal drug distributions without histological sectioning, and label-free microvascular imaging based on endogenous contrast of haemoglobin. For all these applications, sensitivity is orders of magnitude higher than for spontaneous emission or absorption contrast, permitting nonfluorescent reporters for molecular imaging.
Asunto(s)
Microscopía/métodos , Imagen Molecular/métodos , Animales , Oído , Escherichia coli/metabolismo , Fluorescencia , Perfilación de la Expresión Génica , Genes Reporteros/genética , Hemoglobinas/análisis , Carmin de Índigo , Indoles/metabolismo , Operón Lac/genética , Rayos Láser , Ratones , Fármacos Fotosensibilizantes/análisis , Sensibilidad y EspecificidadRESUMEN
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.
Asunto(s)
Microscopía/métodos , Oscilometría/métodos , Espectrometría Raman/métodos , Animales , Diseño de Equipo , Cabello , Rayos Láser , Luz , Ratones , Minoxidil/farmacología , Óptica y Fotónica , Física/métodos , Dispersión de Radiación , Piel/patología , Factores de TiempoRESUMEN
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.
Asunto(s)
Microscopía/instrumentación , Microscopía/métodos , Animales , Rayos Láser , Ratones , Piel/citología , Piel/metabolismo , Espectrometría Raman , Temperatura , Factores de TiempoRESUMEN
Fluorescence microscopy has been widely used to explore the nanoscale world because of its superb sensitivity, but it is limited to fluorescent samples. Hence, various spectroscopic contrasts have been explored for imaging nonfluorescent species. Here we report a multiphoton microscopy based on single-beam near-degenerate four wave mixing (ND-FWM), by detecting a coherent signal generated by the sample at frequencies close to the "edge" of the spectrally "truncated" incident femtosecond pulses. ND-FWM microscopy allows label-free biomedical imaging with high sensitivity and spatial resolution. In particular, by achieving a nearly perfect phase matching condition, ND-FWM generates almost the highest nonlinear coherent signal in a bulk medium and provides a contrast mechanism different from other nonlinear imaging techniques. More importantly, we developed an electronic resonant version of ND-FWM for absorbing but nonfluorescent molecules. Ultrasensitive chromophore detection (approximately 50 molecules) and hemoglobin imaging are demonstrated, by harnessing a fully (triply) resonant enhancement of the nonlinear polarization and using optical heterodyne detection.
Asunto(s)
Microscopía/métodos , Nanotecnología/métodos , Fotones , Análisis Espectral/métodos , Línea Celular Tumoral , Hemoglobinas/química , HumanosRESUMEN
Fluorescence-free microscopy: A new nonlinear optical microspectroscopy technique, femtosecond (fs) triple-resonance coherent anti-Stokes Raman scattering, in which the amplitude and phase of input fs laser pulses are optimally shaped to be in triple resonance with the molecular electronic and vibrational transitions, generates a coherent nonlinear signal beam at a new color with a highest possible efficiency (see figure).
RESUMEN
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.
Asunto(s)
Imagenología Tridimensional/métodos , Lípidos/análisis , Microscopía/métodos , Espectrometría Raman , Animales , Línea Celular Tumoral , Cuerpo Calloso/química , Cuerpo Calloso/citología , Dimetilsulfóxido/administración & dosificación , Dimetilsulfóxido/farmacocinética , Ácido Eicosapentaenoico/metabolismo , Epidermis/química , Epidermis/metabolismo , Epidermis/ultraestructura , Humanos , Ratones , Neuronas/ultraestructura , Sensibilidad y Especificidad , Piel/química , Piel/ultraestructura , Tretinoina/administración & dosificación , Tretinoina/farmacocinética , Vitamina A/análisis , Vitamina A/químicaRESUMEN
Mode-locked operation of a simple Yb:KGW (potassium gadolinium tungstate) oscillator is described, providing 10 W at 1039 nm with a 290 fs pulse width. A polarization-coupled scheme is used for efficient longitudinal pumping by a pair of reshaped laser diode bars. With changes in cavity dispersion, the pulse width is adjustable from 134 to 433 fs, in a high-quality circular mode. A saturable absorber mirror provides self-starting operation, and the cavity is stabilized by the Kerr-lens effect.
RESUMEN
Microscopic imaging of cells and tissues are generated by the interaction of light with either the sample itself or contrast agents that label the sample. Most contrast agents, however, alter the cell in order to introduce molecular labels, complicating live cell imaging. The interaction of light from multiple laser sources has given rise to microscopy, based on Raman scattering or vibrational resonance, which demonstrates selectivity to specific chemical bonds while imaging unmodified live cells. Here, we discuss the nonlinear optical technique of coherent anti-Stokes Raman scattering (CARS) microscopy, its instrumentation, and its status in live cell imaging.
Asunto(s)
Microscopía Confocal/métodos , Microscopía Fluorescente/métodos , Espectrometría Raman/métodos , Animales , Humanos , Citometría de Imagen/instrumentación , Citometría de Imagen/métodos , Citometría de Imagen/tendencias , Rayos Láser , Luz , Microscopía Confocal/instrumentación , Microscopía Confocal/tendencias , Microscopía Fluorescente/instrumentación , Microscopía Fluorescente/tendencias , Óptica y Fotónica , Espectrometría Raman/instrumentación , VibraciónRESUMEN
We induced apoptosis and necrosis in monolayer cultures of Chinese hamster ovary cells using okadaic acid and hydrogen peroxide (H2O2), respectively, and examined the effect on water diffusion and compartmentalization using pulsed-field-gradient (PFG) 1H-NMR and simultaneous confocal microscopy. In PFG experiments characterized by a fixed diffusion time (<4.7 ms) and variable b-values (0-27000 s/mm2), 1H-NMR data collected with untreated cells exhibited multiexponential behavior. Analysis with a slow-exchange model revealed two distinct cellular water compartments with different apparent diffusion coefficients (ADCs; 0.56, 0.06 x 10(-3) mm2/s) and volume fractions (0.96 and 0.04). During the first 12 hr of necrosis or apoptosis, the amount of water in the smallest compartment increased twofold before significant changes in cell density or plasma membrane integrity occurred. Over the same period, water content in the largest compartment decreased by a factor of >2 in apoptotic cells, in accordance with observed cell shrinkage, and changed little in necrotic counterparts, where only slight swelling was evident. These results indicate that PFG 1H-NMR serves as a sensitive indicator of early cell death in monolayer cultures, and can be used to distinguish apoptosis from necrosis. Measurements of restricted diffusion and water exchange are presented to elucidate the compartment origins and justify the model assumptions.