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In fatty acid photodecarboxylase (FAP), light-induced formation of the primary radical product RCOOâ from fatty acid RCOO- occurs in 300â ps, upon which CO2 is released quasi-immediately. Based on the hypothesis that aliphatic RCOOâ (spectroscopically uncharacterized because unstable) absorbs in the red similarly to aromatic carbonyloxy radicals such as 2,6-dichlorobenzoyloxy radical (DCBâ ), much longer-lived linear RCOOâ has been suggested recently. We performed quantum chemical reaction pathway and spectral calculations. These calculations are in line with the experimental DCBâ decarboxylation dynamics and spectral properties and show that in contrast to DCBâ , aliphatic RCOOâ radicals a) decarboxylate with a very low energetic barrier and on the timescale of a few ps and b) exhibit little red absorption. A time-resolved infrared spectroscopy experiment confirms very rapid, âª300â ps RCOOâ decarboxylation in FAP. We argue that this property is required for the observed high quantum yield of hydrocarbons formation by FAP.
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We present a rapid-scanning approach to fluorescence-detected two-dimensional electronic spectroscopy that combines acousto-optic phase-modulation with digital lock-in detection. This approach shifts the signal detection window to suppress 1/f laser noise and enables interferometric tracking of the time delays to allow for correction of spectral phase distortions and accurate phasing of the data. This use of digital lock-in detection enables acquisition of linear and nonlinear signals of interest in a single measurement. We demonstrate the method on a laser dye, measuring the linear fluorescence excitation spectrum as well as rephasing, non-rephasing, and absorptive fluorescence-detected two-dimensional electronic spectra.
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Arbitrary-Detuning ASynchronous OPtical Sampling (ADASOPS) is a pump-probe technique which relies on the stability of femtosecond oscillators. It provides access to a multiscale time window ranging up to millisecond, combined with a sub-picosecond time resolution. In contrast with the first ADASOPS demonstration based on the interferometric detection of coincidences between optical pulses, we show here that the optical setup can now be reduced to a mere pair of photodetectors embedded in a specially-designed electronic system. In analogy with super-resolution methods used in optical microscopy for localizing single emitters beyond the diffraction limit, we demonstrate that purely electronic means allow the determination of time delays between each pump-probe pulse pair with a standard deviation as small as 200 fs. The new method is shown to be simpler, more versatile and more accurate than the coincidence-based approach.
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Femtosecond amplifiers seeded by two independent femtosecond oscillators normally produce amplified pulse pairs with a timing jitter equal to the oscillator period, which is typically around 12 ns for Titanium:Sapphire lasers. Combining Arbitrary-Detuning Asynchronous Optical Sampling (AD-ASOPS) with an appropriate selection of amplified pulses, we demonstrate that the time-delay distribution can be narrowed down to a 25-ps time window, allowing to produce spectral interference fringes for each amplified pulse pair. Subsequent AD-ASOPS determination of the actual time delay with subpicosecond accuracy allows to tailor the delay distribution with an electronic control all the way to the repetition period of the amplifiers. We thus demonstrate rapid scanning of the time delays up to nearly 1 ms with a sub-picosecond accuracy, which makes this method an ideal tool for multiscale pump-probe spectroscopy.
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We demonstrate that Arbitrary-Detuning ASynchronous OPtical Sampling (AD-ASOPS) makes possible multiscale pump-probe spectroscopy with time delays spanning from picosecond to millisecond. The implementation on pre-existing femtosecond amplifiers seeded by independent free-running oscillators is shown to be straightforward. The accuracy of the method is determined by comparison with spectral interferometry, providing a distribution with a standard deviation ranging from 0.31 to 1.7 ps depending on experimental conditions and on the method used to compute the AD-ASOPS delays.
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A recently reported variant of asynchronous optical sampling compatible with arbitrary unstabilized laser repetition rates is applied to pump-probe spectroscopy. This makes possible the use of a 5.1 MHz chirped pulse oscillator as the pump laser, thus extending the available time window to almost 200 ns with a time resolution as good as about 320 fs. The method is illustrated with the measurement in a single experiment of the complete charge transfer dynamics of the reaction center from Rhodobacter sphaeroides.
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Dispositivos Ópticos , Proteínas del Complejo del Centro de Reacción Fotosintética/metabolismo , Rhodobacter sphaeroides/metabolismo , Análisis Espectral , Transporte de ElectrónRESUMEN
A method of asynchronous optical sampling based on free-running lasers with no requirement on the repetition rates is presented. The method is based on the a posteriori determination of the delay between each pair of pulses. A resolution better than 400 fs over 13 ns total delay scan is demonstrated. In addition to the advantages of conventional asynchronous sampling techniques, this method allows a straightforward implementation on already-existing laser systems using a fiber-based setup and an appropriate acquisition procedure.
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Rayos Láser , Fenómenos Ópticos , Interferometría , Probabilidad , Procesamiento de Señales Asistido por Computador , Factores de TiempoRESUMEN
Solar ultraviolet longwave UVA1 exposure of human skin has short-term consequences at cellular and molecular level, leading at long-term to photoaging. Following exposure, reactive oxygen species (ROS) are generated, inducing oxidative stress that might impair cellular metabolic activity. However, the dynamic of UVA1 impact on cellular metabolism remains unknown because of lacking adequate live imaging techniques. Here we assess the UVA1-induced metabolic stress response in reconstructed human skin with multicolor two-photon fluorescence lifetime microscopy (FLIM). Simultaneous imaging of nicotinamide adenine dinucleotide (NAD(P)H) and flavin adenine dinucleotide (FAD) by wavelength mixing allows quantifying cellular metabolism in function of NAD(P)+/NAD(P)H and FAD/FADH2 redox ratios. After UVA1 exposure, we observe an increase of fraction of bound NAD(P)H and decrease of fraction of bound FAD indicating a metabolic switch from glycolysis to oxidative phosphorylation or oxidative stress possibly correlated to ROS generation. NAD(P)H and FAD biomarkers have unique temporal dynamic and sensitivity to skin cell types and UVA1 dose. While the FAD biomarker is UVA1 dose-dependent in keratinocytes, the NAD(P)H biomarker shows no dose dependence in keratinocytes, but is directly affected after exposure in fibroblasts, thus reflecting different skin cells sensitivities to oxidative stress. Finally, we show that a sunscreen including a UVA1 filter prevents UVA1 metabolic stress response from occurring.
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Flavina-Adenina Dinucleótido/metabolismo , NADP/metabolismo , Piel/metabolismo , Piel/efectos de la radiación , Estrés Fisiológico/efectos de la radiación , Rayos Ultravioleta , Biomarcadores , Aprendizaje Profundo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Microscopía Fluorescente , Imagen Óptica , Luz SolarRESUMEN
We demonstrate selective two-photon excited fluorescence microscopy with shaped pulses produced with a simple yet efficient scheme based on dispersive optical components. The pulse train from a broadband oscillator is split into two subtrains that are sent through different amounts of glass. Beam recombination results in pulse-shape switching at a rate of 150MHz. Time-resolved photon counting detection then provides two simultaneous images resulting from selective two-photon excitation, as demonstrated in a live embryo. Although less versatile than programmable pulse-shaping devices, this novel arrangement significantly improves the performance of selective microscopy using broadband shaped pulses while simplifying the experimental setup.
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The mechanical properties of biological tissues are strongly correlated to the specific distribution of their collagen fibers. Monitoring the dynamic reorganization of the collagen network during mechanical stretching is however a technical challenge, because it requires mapping orientation of collagen fibers in a thick and deforming sample. In this work, a fast polarization-resolved second harmonic generation microscope is implemented to map collagen orientation during mechanical assays. This system is based on line-to-line switching of polarization using an electro-optical modulator and works in epi-detection geometry. After proper calibration, it successfully highlights the collagen dynamic alignment along the traction direction in ex vivo murine skin dermis. This microstructure reorganization is quantified by the entropy of the collagen orientation distribution as a function of the stretch ratio. It exhibits a linear behavior, whose slope is measured with a good accuracy. This approach can be generalized to probe a variety of dynamic processes in thick tissues.
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Colágeno/metabolismo , Fenómenos Mecánicos , Microscopía , Piel/diagnóstico por imagen , Piel/metabolismo , Animales , Fenómenos Biomecánicos , Procesamiento de Imagen Asistido por Computador , Ratones , Factores de TiempoRESUMEN
Affiliation 4 incorrectly read 'University of the Basque Country (Ikerbasque), University of the Basque Country and Donostia International Physics Center, San Sebastian 20018, Spain.'Also, the affiliations of Ignacio Arganda-Carreras with 'IKERBASQUE, Basque Foundation for Science, Bilbao, 48013, Spain' and 'Donostia International Physics Center (DIPC), San Sebastian, 20018, Spain' were inadvertently omitted.Additionally, the third sentence of the first paragraph of the Results section entitled 'Multicontrast organ-scale imaging with ChroMS microscopy' incorrectly read 'For example, one can choose lambda1 = 850 and lambda2 = 110 nm for optimal two-photon excitation of blue and red chromophores.'. The correct version reads 'lambda2 = 1100 nm' instead of 'lambda2 = 110 nm'. These errors have now been corrected in the PDF and HTML versions of the Article.
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Large-scale microscopy approaches are transforming brain imaging, but currently lack efficient multicolor contrast modalities. We introduce chromatic multiphoton serial (ChroMS) microscopy, a method integrating one-shot multicolor multiphoton excitation through wavelength mixing and serial block-face image acquisition. This approach provides organ-scale micrometric imaging of spectrally distinct fluorescent proteins and label-free nonlinear signals with constant micrometer-scale resolution and sub-micron channel registration over the entire imaged volume. We demonstrate tridimensional (3D) multicolor imaging over several cubic millimeters as well as brain-wide serial 2D multichannel imaging. We illustrate the strengths of this method through color-based 3D analysis of astrocyte morphology and contacts in the mouse cerebral cortex, tracing of individual pyramidal neurons within densely Brainbow-labeled tissue, and multiplexed whole-brain mapping of axonal projections labeled with spectrally distinct tracers. ChroMS will be an asset for multiscale and system-level studies in neuroscience and beyond.
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Corteza Cerebral/diagnóstico por imagen , Imagenología Tridimensional/métodos , Proteínas Luminiscentes/química , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , Neuroimagen/métodos , Animales , Astrocitos/metabolismo , Corteza Cerebral/citología , Color , Dependovirus , Femenino , Vectores Genéticos/administración & dosificación , Vectores Genéticos/genética , Células HEK293 , Humanos , Proteínas Luminiscentes/genética , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Modelos Animales , Nestina/genética , Técnicas de Trazados de Vías Neuroanatómicas/métodos , Parvovirinae/genética , Células Piramidales/metabolismo , TransfecciónRESUMEN
Two-photon imaging of endogenous fluorescence can provide physiological and metabolic information from intact tissues. However, simultaneous imaging of multiple intrinsic fluorophores, such as nicotinamide adenine dinucleotide(phosphate) (NAD(P)H), flavin adenine dinucleotide (FAD) and retinoids in living systems is generally hampered by sequential multi-wavelength excitation resulting in motion artifacts. Here, we report on efficient and simultaneous multicolor two-photon excitation of endogenous fluorophores with absorption spectra spanning the 750-1040 nm range, using wavelength mixing. By using two synchronized pulse trains at 760 and 1041 nm, an additional equivalent two-photon excitation wavelength at 879 nm is generated, and achieves simultaneous excitation of blue, green and red intrinsic fluorophores. This method permits an efficient simultaneous imaging of the metabolic coenzymes NADH and FAD to be implemented with perfect image co-registration, overcoming the difficulties associated with differences in absorption spectra and disparity in concentration. We demonstrate ratiometric redox imaging free of motion artifacts and simultaneous two-photon fluorescence lifetime imaging (FLIM) of NADH and FAD in living tissues. The lifetime gradients of NADH and FAD associated with different cellular metabolic and differentiation states in reconstructed human skin and in the germline of live C. Elegans are thus simultaneously measured. Finally, we present multicolor imaging of endogenous fluorophores and second harmonic generation (SHG) signals during the early stages of Zebrafish embryo development, evidencing fluorescence spectral changes associated with development.
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Caenorhabditis elegans/metabolismo , Fibroblastos/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Microscopía de Fluorescencia por Excitación Multifotónica/métodos , NADP/metabolismo , Retinoides/metabolismo , Piel/metabolismo , Animales , Caenorhabditis elegans/citología , Fibroblastos/citología , Humanos , Piel/citologíaRESUMEN
We demonstrate selective fluorescence We demonstrate selective fluorescence excitation of specific molecular species in live organisms by using coherent control of two-photon excitation. We have acquired quasi-simultaneous images in live fluorescently-labeled Drosophila embryos by rapid switching between appropriate pulse shapes. Linear combinations of these images demonstrate that a high degree of fluorophore selectivity is attainable through phase-shaping. Broadband phase-shaped excitation opens up new possibilities for single-laser, multiplex, in-vivo fluorescence microscopy.
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The use of a fast temperature jump (T-jump) is a very powerful experiment aiming at studying protein denaturation dynamics. However, probing the secondary structure is a difficult challenge and rarely yields quantitative values. We present the technical implementation of far-UV circular dichroism in a nanosecond T-jump experiment and show that this experiment allows us to follow quantitatively the change in the helical fraction of a poly(glutamic acid) peptide during its thermal denaturation with 12 ns time resolution.
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Dicroismo Circular/métodos , Temperatura , Absorción , Dicroismo Circular/instrumentación , Rayos Láser , Ácido Poliglutámico/química , Desnaturalización Proteica , Espectrofotometría Infrarroja , Espectrofotometría Ultravioleta , Factores de TiempoRESUMEN
Quantifying cell behaviors in animal early embryogenesis remains a challenging issue requiring in toto imaging and automated image analysis. We designed a framework for imaging and reconstructing unstained whole zebrafish embryos for their first 10 cell division cycles and report measurements along the cell lineage with micrometer spatial resolution and minute temporal accuracy. Point-scanning multiphoton excitation optimized to preferentially probe the innermost regions of the embryo provided intrinsic signals highlighting all mitotic spindles and cell boundaries. Automated image analysis revealed the phenomenology of cell proliferation. Blastomeres continuously drift out of synchrony. After the 32-cell stage, the cell cycle lengthens according to cell radial position, leading to apparent division waves. Progressive amplification of this process is the rule, contrasting with classical descriptions of abrupt changes in the system dynamics.