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The limitations of 2D microscopy constrain our ability to observe and understand tissue-wide networks that are, by nature, 3-dimensional. Optical projection tomography enables the acquisition of large volumes (ranging from micrometres to centimetres) in various tissues, with label-free capacities for the observation of auto-fluorescent signals as well fluorescent-labelled targets of interest in multiple channels. We present a multi-modal workflow for the characterization of both structural and quantitative parameters of the mouse small intestine. As proof of principle, we evidence its applicability for imaging the mouse intestinal immune compartment and surrounding mucosal structures. We quantify the volumetric size and spatial distribution of Isolated Lymphoid Follicles (ILFs) and quantify density of villi throughout centimetre long segments of intestine. Furthermore, we exhibit the age- and microbiota-dependence for ILF development, and leverage a technique that we call reverse-OPT for identifying and homing in on regions of interest. Several quantification capabilities are displayed, including villous density in the autofluorescent channel and the size and spatial distribution of the signal of interest at millimetre-scale volumes. The concatenation of 3D image acquisition with the reverse-OPT sample preparation and a 2D high-resolution imaging modality adds value to interpretations made in 3D. This cross-modality referencing technique is found to provide accurate localisation of ROIs and to add value to interpretations made in 3D. Importantly, OPT may be used to identify sparsely-distributed regions of interest in large volumes whilst retaining compatibility with high-resolution microscopy modalities, including confocal microscopy. We believe this pipeline to be approachable for a wide-range of specialties, and to provide a new method for characterisation of the mouse intestinal immune compartment.

Vision in living mice is sensitive to femtosecond pulse shaping., Vision is usually assumed to be sensitive to the light intensity and spectrum but not to its spectral phase. However, experiments performed on retinal proteins in solution showed that the first step of vision consists in an ultrafast photoisomerization that can be coherently controlled by shaping the phase of femtosecond laser pulses, especially in the multiphoton interaction regime. The link between these experiments in solution and the biological process allowing vision was not demonstrated. Here, we measure the electric signals fired from the retina of living mice upon femtosecond multipulse and single-pulse light stimulation. Our results show that the electrophysiological signaling is sensitive to the manipulation of the light excitation on a femtosecond time scale. The mechanism relies on multiple interactions with the light pulses close to the conical intersection, like pump-dump (photoisomerization interruption) and pump-repump (reverse isomerization) processes. This interpretation is supported both experimentally and by dynamics simulations.

Optical projection tomography (OPT) is a powerful tool for three-dimensional imaging of mesoscopic biological samples with great use for biomedical phenotyping studies. We present a fluorescent OPT platform that enables direct visualization of biological specimens and processes at a centimeter scale with high spatial resolution, as well as fast data throughput and reconstruction. We demonstrate nearly isotropic sub-28 µm resolution over more than 60 mm3 after reconstruction of a single acquisition. Our setup is optimized for imaging the mouse gut at multiple wavelengths. Thanks to a new sample preparation protocol specifically developed for gut specimens, we can observe the spatial arrangement of the intestinal villi and the vasculature network of a 3-cm long healthy mouse gut. Besides the blood vessel network surrounding the gastrointestinal tract, we observe traces of vasculature at the villi ends close to the lumen. The combination of rapid acquisition and a large field of view with high spatial resolution in 3D mesoscopic imaging holds an invaluable potential for gastrointestinal pathology research.

We demonstrate femtosecond time–resolved soft–X–ray absorption spectroscopy of liquid samples by combining a sub–micrometer–thin flat liquid jet with a high–harmonic table–top source covering the entire water-window range (284 - 538 eV). Our measurements temporally resolve the gradual appearance of absorption features below the carbon K-edge of ethanol and methanol during strong–field ionization, which trace the valence-shell ionization dynamics of the liquid alcohols with a temporal resolution of ~30 fs.

This chapter presents TR-XAS measurement results in the liquid phase. This chapter presents TR-XAS measurement results in the liquid phase. By coupling a vacuum flat jet interaction chamber with the HHG water window source described in Chap. 2 and taking advantage of the cut-off extension presented in Chap. 4, the induced reaction is tracked simultaneously from the carbon and nitrogen K-edges at sub-100 fs time resolution. Measurements were performed on the liquid pyridine (C5H5N) excited by ultrashort NIR pulses in the high intensity regime and transient X-ray absorption near-edge structure (XANES) modifications were observed.

Time-resolved X-ray absorption spectroscopy has for long been experimentally limited to picosecond temporal resolution measurements in large-scale facilities. Recently, the expansion of high-harmonic generation table-top laser sources have led the femtosecond resolution of Ti:Sa oscillators to measurements in the extreme ultraviolet. This experimental chapter details the first results of the transient absorption setup developed in collaboration with the group of H.J. Worner of ETHZ that are described in Chap. 2. This experiment measures the dissociation of molecules in the gas phase from the sulfur L-edges and the carbon K-edge in the soft X-rays. The temporal resolution of the setup allows to follow light-induced chemical reactions of the lowest electronic states with a few femtoseconds resolution. By comparing the calculated core-level transitions with the observed splitting of orbital states, the assignment of resolved structures, in accordance with the literature, allows to reconstruct the geometry changes of the molecules along their reaction path.

This chapter presents the HHG driving pulse optimization results. The intensity and short pulse requirements to reach higher soft X-ray energies and better conversion ratio are discussed and different approaches are introduced. The previous, demanding, time-resolved X-ray absorption spectroscopy experiment in the gas phase leaned towards the option for an easy pulse shortening scheme. The adopted two-stage pulse broadening by filamentation coupled to a very simple post-compression technique is environmentally robust and leads to a 0.2 TW sub-two-cycle short-wavelength infrared pulse. The latter can drive high-order harmonics beyond the oxygen K absorption edge with similar flux conditions as obtained during the successful gas dissociation measurements at the carbon K-edge.

This chapter introduces in detail the different elements assembled and designed to get the soft X-ray transient absorption experiment ready. Some maintenance routines and operational specifications naturally stem from the laser source’s reliance on a commercial system. It describes and details every pulse involved in the experiment, as well as the optical post-treatments applied to the pump pulse and the high harmonic generation driver to increase the system overall performances. The condition of soft X-ray generation in helium with long wavelength lasers is discussed and compared to the most advanced similar systems, providing an overview of the state of the art for tabletop time-resolved absorption spectroscopy in the water window. The characteristics of the flat-field vacuum spectrometers that were used to measure the signals over the full water window are also presented and discussed in detail. Finally, the data acquisition and data analysis methods are provided.

Femtosecond X-ray absorption spectroscopy (XAS) is a powerful method to investigate the dynamical behavior of a system after photoabsorption in real time. So far, the application of this technique has remained limited to large-scale facilities, such as femtosliced synchrotrons and free-electron lasers (FEL). In this work, we demonstrate femtosecond time-resolved soft-X-ray absorption spectroscopy of liquid samples by combining a sub-micrometer-thin flat liquid jet with a high-harmonic tabletop source covering the entire water-window range (284–538 eV). Our work represents the first extension of tabletop XAS to the oxygen edge of a chemical sample in the liquid phase. In the time domain, our measurements resolve the gradual appearance of absorption features below the carbon K-edge of ethanol and methanol during strong-field ionization and trace the valence-shell ionization dynamics of the liquid alcohols with a temporal resolution of ∼30 fs. This technique opens unique opportunities to study molecular dynamics of chemical systems in the liquid phase with elemental, orbital, and site sensitivity. ISSN:1948-7185

We demonstrate femtosecond time–resolved soft–X–ray absorption spectroscopy of liquid samples by combining a sub–micrometer–thin flat liquid jet with a high–harmonic table–top source covering the entire water-window range (284 - 538 eV). Our work represents the first extension of table-top XAS to the oxygen edge of a chemical sample in the liquid phase. In the time domain, our measurements resolve the gradual appearance of absorption features below the carbon K-edge of ethanol and methanol during strong–field ionization, which trace the valence-shell ionization dynamics of the liquid alcohols with a temporal resolution of ~30 fs.

Science, 355 (6322), ISSN:0036-8075, ISSN:1095-9203

This work studies the relaxation dynamics of molecules in both the gas and liquid phases after strong field ionization, using transient absorption in the soft X-rays. In particular, the thesis presents the first realization of time-resolved X-ray absorption spectroscopy in the spectral water window with a laser-based HHG source. These remarkable experiments were not only performed for isolated molecules, but also in liquids, for which the spectral coverage of the K-edges of C, N, and O are of primary importance for investigating biological molecules. The technique relies on the generation of high-order harmonics to further probe the electronic structure of molecules. Using the atomic selectivity of high energies and the temporal coherence of laser technology, we demonstrate the observation of the first stages of chemical transformation of matter in the gas and liquid phases.

We demonstrate the simultaneous generation of second, third, and fourth harmonic from a single dielectric Bismuth Ferrite nanoparticle excited by a telecom fiber laser at 1560 nm. We first characterize the signals associated with different nonlinear orders in terms of spectrum, excitation intensity dependence, and relative signal strengths. Successively, on the basis of the polarization-resolved emission curves of the three harmonics, we discuss the interplay of susceptibility tensor components at the different orders and we show how polarization can be used as an optical handle to control the relative frequency conversion properties., Comment: include S.I. section at the end of the main pdf file

Optics Express, 26 (9), ISSN:1094-4087

Time-resolved spectroscopy at element-specific absorption edges provides a powerful tool to study ultrafast phenomena of matter on the atomic scale in the gas, solid or liquid phase. Combining element sensitivity of core-to-valence transitions due to the very localized initial core state and femtosecond/attosecond time resolution of new soft X-ray sources based on high-harmonic generation from long-wavelength drivers, accurate electronic and structural information is now accessible.

We use Hyper Rayleigh Scattering and polarization resolved multiphoton microscopy to investigate simultaneously the second and third-order nonlinear response of Potassium Niobate and Bismuth Ferrite harmonic nanoparticles. We first derive the second-to-third harmonic intensity ratio for colloidal ensembles and estimate the average third-order efficiency of these two materials. Successively, we explore the orientation dependent tensorial response of individual nanoparticles fixed on a substrate. The multi-order polarization resolved emission curves are globally fitted with an analytical model to retrieve individual elements of susceptibility tensors.

This review provides a comprehensive overview of the structural dynamics in topical gas- and condensed-phase systems on multiple length and time scales. Starting from vibrationally induced dissociation of small molecules in the gas phase, the question of vibrational and internal energy redistribution through conformational dynamics is further developed by considering coupled electron/proton transfer in a model peptide over many orders of magnitude. The influence of the surrounding solvent is probed for electron transfer to the solvent in hydrated I−. Next, the dynamics of a modified PDZ domain over many time scales is analyzed following activation of a photoswitch. The hydration dynamics around halogenated amino acid side chains and their structural dynamics in proteins are relevant for iodinated TyrB26 insulin. Binding of nitric oxide to myoglobin is a process for which experimental and computational analyses have converged to a common view which connects rebinding time scales and the underlying dynamics. Finally, rhodopsin is a paradigmatic system for multiple length- and time-scale processes for which experimental and computational methods provide valuable insights into the functional dynamics. The systems discussed here highlight that for a comprehensive understanding of how structure, flexibility, energetics, and dynamics contribute to functional dynamics, experimental studies in multiple wavelength regions and computational studies including quantum, classical, and more coarse grained levels are required.