Simultaneous field- and aperture-plane (back-focal plane, BFP) imaging enriches the information content of fluorescence microscopy. In addition to the usual density and concentration maps of sample-plane images, BFP images provide information on the surface proximity and orientation of molecular fluorophores. They also give access to the refractive index of the fluorophore-embedding medium. However, in the high-NA, wide-field detection geometry commonly used in single-molecule localisation microscopies, such measurements are averaged over all fluorophores present in the objective’s field of view, thus limiting spatial resolution and specificity. We here solve this problem and demonstrate how an oblique, variable-angle, coherent ring illumination can be used to generate a Bessel beam that - for supercritical excitation angles - produces an evanescent needle of light. Scanning the sample through the this evanescent needle enables us to acquire combined sample-plane and BFP images with sub-diffraction resolution and axial localisation precision. Background, resolution and polarisation considerations will be discussed.

Total internal reflection microscopy is a technique ideal for imaging the cell membrane, as it allows for selective excitation of fluorophores at or near the coverglass. When a beam enters a high NA objective off-axis, it produces an evanescent field, the penetration depth of which is dependent on the refractive index difference between the glass substrate and sample. A pitfall of this method is that sample refractive index varies across a cell surface and organelles, causing unwanted light refraction and scattering. By introducing a Bertrand lens into the detection optics, the critical angle on the back focal plane can be imaged, which allows to calculate the sample refractive angle, and emission from near-membrane and deeper fluorophores can be separated, too. Here, we use a scanning TIR-SAF system to measure the local cellular refractive index across the cell's “footprint” region. The excitation path contains a radial polarizer, and two axicon lenses to form evanescent Bessel beam illumination, allowing for an excitation “needle”. Tightly focused radially and azimuthally polarized Bessel beams are desirable as they allow for a smaller excitation spot size. However, neither effects of azimuthally nor radially polarized excitation on evanescent field formation have been characterized. We built and characterized a confocal TIR-SAF system, and the effects of polarization were assessed.

Wide-field imaging conventionally results in a single image plane oriented perpendicular to the optical axis. However, in brain slice or in vivo recording, neuronal or circuit morphologies lie in arbitrarily tilted planes. Consequently the spatiotemporal advantages of wide-field non-scanned imaging are lost because of the time required for stepwise focal readjustments to view an entire neuron or network. We describe an application of remote focus that views simultaneously two planes separated by up to 100 µm, each with variable tilt from the conventional image plane. This permits fluorescence detection of ion fluxes or membrane potential across neuronal compartments and their correlation with electrical activity. Further, two fluorophores can be viewed simultaneously in each plane. We show (i) neuronal images tilted to optimise simultaneous aquisition of somatic, dendritic and axonal compartments; (ii) networks viewed simultaneously at 2 depths separated by up to 100 µm, (iii) widefield imaging at 30 Hz of Gcamp5 fluorescence during spontaneous spiking in motoneuron layers of zebrafish spinal cord separated by 30-40 microns.

Ultraviolet (UV) synchrotron radiation circular dichroism(SRCD) spectroscopy has made an important contribution to the determination andunderstanding of the structure of bio-molecules. In this paper, we report aninnovative approach that we term time-resolvedSRCD (tr-SRCD), which overcomes the limitations of current broadband UV SRCDsetups. This technique allows accessing ultrafast time scales(down to nanoseconds), previously measurable only by other methods, such asinfrared (IR), nuclear magnetic resonance (NMR), fluorescence and absorbancespectroscopies and small angle X-ray scattering (SAXS). The tr-SRCD setup takesadvantage of the natural polarisation of the synchrotron radiation emitted by abending magnet to record broadband UV CD faster than any current SRCD setup,improving the acquisition speed from 10 mHz to 130 Hz and the accessibletemporal resolution by several orders of magnitude. We illustrate the newapproach by following the isomers concentration changes of an azopeptide aftera photoisomerisation. This breakthrough in SRCD spectroscopy opens up a widerange of potential applications to the detailed characterisation of biologicalprocesses, such as protein folding, protein-ligand binding.

The DEIMOS (Dichroism Experimental Installation for Magneto-Optical Spectroscopy) beamline was part of the second phase of the beamline development at French Synchrotron SOLEIL (Source Optimisée de Lumière à Energie Intermédiaire du LURE) and opened to users in March 2011. It delivers polarized soft x-rays to perform x-ray absorption spectroscopy, x-ray magnetic circular dichroism, and x-ray linear dichroism in the energy range 350-2500 eV. The beamline has been optimized for stability and reproducibility in terms of photon flux and photon energy. The main end-station consists in a cryo-magnet with 2 split coils providing a 7 T magnetic field along the beam or 2 T perpendicular to the beam with a controllable temperature on the sample from 370 K down to 1.5 K.

An alternate multilayer (AML) grating has been prepared by coating an ion etched lamellar grating with a B4C/Mo2C multilayer (ML) having a layer thickness close to the groove depth. Such a structure behaves as a 2D synthetic crystal and can reach very high efficiencies when the Bragg condition is satisfied. This AML coated grating has been characterized at the SOLEIL Metrology and Tests Beamline between 0.7 and 1.7 keV and at the four-crystal monochromator beamline of Physikalisch-Technische Bundesanstalt (PTB) at BESSY II between 1.75 and 3.4 keV. A peak diffraction efficiency of nearly 27% was measured at 2.2 keV. The measured efficiencies are well reproduced by numerical simulations made with the electromagnetic propagation code CARPEM. Such AML gratings, paired with a matched ML mirror, constitute efficient monochromators for intermediate energy photons. They will extend the accessible energy for many applications as x-ray absorption spectroscopy or x-ray magnetic circular dichroism experiments.

DESIRS is a new undulator-based VUV beamline at SOLEIL (France) optimized for the study of gas phase matter in the 5-40 eV range. It is equipped with two dedicated endstations: a VUV Fourier-Transform Spectrometer (FTS) for ultra-high resolution absorption spectroscopy (resolving power up to 106) and an electron/ion imaging coincidence spectrometer. The photon source is a 10 m-long pure electromagnetic variable polarization undulator providing, at the sample location, fully calibrated quasi-perfect horizontal, vertical and circular polarizations. The optical design includes a beam waist allowing the implementation of a gas filter to suppress the undulator higher harmonics. The 6.65 m Eagle off-plane Normal Incidence Monochromator equipped with four gratings allows the tuning of the flux-to-resolution trade-off. Measured ultimate instrumental resolving powers are 124000 (174 μeV) around 21 eV and 250000 (54 μeV) around 13 eV, while the typical measured flux is in the 1010−1011 ph/sec range in a 1/50000 bandwidth and 1012-1013 ph/sec in a 1/1000 bandwidth.

SEXTANTS is a new SOLEIL beamline dedicated to soft X-ray scattering techniques. The beamline, covering the 50-1700 eV energy range, features two Apple-II undulators for polarization control and a fixed-deviation monochromator. Two branch-lines host three end-stations for elastic, inelastic and coherent scattering experiments.

Multilayer (ML) gratings have been prepared by coating shallow ion etched lamellar gratings with a Mo2C/B4C multilayer having a layer thickness close to the groove depth. It was shown that such a structure behaves as a 2D synthetic crystal and can reach very high efficiencies when the Bragg condition is satisfied. A ML coated grating has been characterized at the SOLEIL Metrology beamline between 700 and 1700 eV and a beamline of PTB at BESSY II between 1750 and 3500 eV. A peak diffraction efficiency of nearly 27 % was measured at 2200 eV. The measured efficiencies are well reproduced by numerical simulations made with the electromagnetic propagation code CARPEM, This grating will be used in the Deimos beamline at SOLEIL together with a matched multilayer mirror.

We present a comparative study of B4C/Mo and B4C/Mo2C periodic multilayer structures deposited by magnetron sputtering. The characterization was performed by grazing incidence X-ray reflectometry at two different energies and high resolution transmission electron microscopy. The experimental results indicate the existence of an interdiffusion layer at the B4C-on-Mo interface in the B4C/Mo system. Thus, the B4C/Mo multilayers were modeled by an asymmetric structure with three layers in each period. The thickness of B4C-on-Mo interfacial layer was estimated about 1.1 nm. The B4C/Mo2C multilayers present less interdiffusion and are well modeled by a symmetric structure without interfacial layers. This study shows that B4C/Mo2C structure is an interesting alternative to B4C/Mo multilayer for X-ray optic applications.

The brightness of a synchrotron source coupled to an infrared microscope allows imaging at the so-called diffraction limit. Thus, numerous infrared beamlines around the world have been developed for infrared chemical imaging. Infrared microscopes employ Schwarzschild objectives, which are based on two spherical mirrors centered on a common optical axis. A common drawback of the latter design comes with the central obscuring of the incident beam and the corresponding diffraction effects. However, in this work we are taking advantage of the "shadow" below the primary mirror in order to "vertically" probe our sample and record depth profiling images. We recorded infrared confocal images of liquid water occluded inside a quartz micro-cavity and reconstructed a 3D infrared image based on the OH stretching vibrational bands. Although only a restricted sample size can be analyzed, we demonstrate here the feasibility of a 3D confocal infrared measurement. The chemical profiles have been calculated along the Z-axis of the water inclusion to study its heterogeneity and water-solid interfaces.

The DISCO beamline at SOLEIL synchrotron radiation facility [1] is a low energy beamline (1-20 eV) with three end-stations. The imaging branch is the lowest energy of the three, ranging between 1 and 5 eV [2], for which ultra high vacuum is not mandatory. Therefore, the branch is separated from the beamline vacuum by a DN63CF suprasyl window protected for colour centre formation by suprasyl plate moveable under vacuum. In order to maximise the reflectivity in the photon energy domain of interest, all the mirrors specific to the branch have been coated with Al-MgF2. Commissioning and first year of exploitation were performed under primary vacuum conditions in the imaging branch line. During that period, the optics got heavily polluted, which induced a strong loss of photon flux. Al-MgF2 optics cannot be cleaned using oxygen plasma without heavy damages and loss of reflectivity. Therefore, the mirrors had to be removed and cleaned ex situ. Following this maintenance, the branch had been re-exploited under high vacuum conditions assured by a turbomolecular pumping to provide what we expected to be better protection of the optical elements. Although the very first photometric measurements showed a gain in flux, very soon the photon flux starts decreasing.

The so called varied groove depth (VGD) etching technology, developed by Horiba-Jobin-Yvon, which consists in laterally grading the groove depth of a lamellar grating, is exploited in the monochromators of the seven soft x-ray beamlines of SOLEIL. With a VGD profile a single grating can perform in more than a decade wide energy band and some control is given over the harmonic content. It also requires a specific, but easily achieved design of the beamline optics to match the lateral footprint of the beam to the depth variation. Performances predicted by computation with electromagnetic propagation code are supported by flux measurements of the two first harmonics in the experimental chamber of a photoemission beamline, and by measurements of grating efficiencies on SOLEIL etrology beamline.

This paper presents a study of B4C/Mo2C multilayers mirrors with the aim of using it in the achievement of Alternate MultiLayer (AML) grating. Such component allows a high efficiency in the 500-2500 eV energy range for the DEIMOS beamline. Multilayers were deposited on silicon substrate. They are characterized by reflectometry under grazing incidence. Numerical adjustments were performed with a model of two layers in the period without any interfacial. A prototype of AML grating was fabricated and characterized. The efficiency of the first order of diffraction was worth 15% at 1700 eV.

As one of the latest beamline built at the SOLEIL synchrotron source, ANTARES beamline offers a spectroscopic non-destructive nano-probe to study advanced materials. This innovative scanning photoemission microscopy combines linear and angle sweeps to perform precise electronic band structure determination by Nano Angle Resolved Photoelectron Spectroscopy (nanoARPES) and chemical imaging by core level detection. The beamline integrates effectively insertion devices and a high transmission beamline optics. This photon source has been combined with an advanced microscope, which has precise sample handling abilities. Moreover, it is fully compatible with a high resolution R4000 Scienta hemispherical analyzer and a set of Fresnel Zone Plates (FZP) able to focalize the beam spot up to a few tenths of nanometers, depending on the spatial resolution of the selected FZP. We present here the main conceptual design of the beamline and endstation, together with some of the firsts commissioning results.

Shaping a YAG scintillator crystal into a truncated-ball lens enables to image with a high numerical aperture its front surface, where the image converted from x-rays to visible is localized. Hence, both resolution and luminosity gains can be expected. Moreover if the plane surface is set at the Young-Weierstrass point of the spherical refractive surface, stigmatic imaging is achieved. On this principle, we have constructed an imaging detector from a 10 mm diameter YAG:Ce sphere and a long working distance plane-apochromatic microscope objective which does not limit the numerical aperture. The effective numerical aperture of the built system is 1.08, giving a Rayleigh resolution limit of 0.3 mu m. Images of test objects (diatoms) have been recorded, in contact mode, with 103 eV. Periodic features of 0.4 mu m pitch are visible on these images. The field of view is close to 200 mu m. The device is intended as an aid for X-ray optics fine-tuning and characterization.

The DEIMOS (Dichroism Experimental Installation for Magneto-Optical Spectroscopy) beamline is a SOLEIL Phase-II beamline and has been opened to users since March 2011. It delivers polarized soft x-rays to perform x-ray absorption spectroscopy, x-ray magnetic circular dichroism and x-ray linear dichroism in the energy range 350-2500 eV. The beamline has been optimized for stability and reproducibility in terms of photon flux and photon energy. The main end-station consists in a cryo-magnet with 2 split coils allowing 7T along the beam or 2T perpendicular to the beam with a variable temperature on the sample from 370K down to 1.5K.

The new synchrotron-radiation circular-dichroism (SRCD) endstation on the UV-visible synchrotron beamline DISCO has been commissioned at the SOLEIL synchrotron. The design has been focused on preservation of a high degree of linear polarization at high flux and moderate resolving power covering the vacuum ultraviolet to visible spectral range (125-600 nm). The beam dimensions have been set to 4 mm × 4 mm at 1 nm bandwidth for lower sample degradation. The nitrogen-purged sample chamber fits three types of sample holders accommodating conventional round cell mounting, automated rotation of the samples, as well as a microfluidic set-up. Automated temperature-controlled data collection on microvolumes is now available to the biology and chemistry communities. Macromolecules including membrane proteins, soluble proteins, bio-nanotubes, sugars, DNA and RNAs are now routinely investigated.

Before the first photon beam was delivered at the SOLEIL synchrotron, scientists tried to anticipate the problem of carbon contamination on optical components, with for instance the outgasing of chambers by prior exposure to the beam with dummy optics. In spite of these efforts, deterioration of optical performance by carbon contamination has remained an outstanding issue: on the low-energy beamlines at SOLEIL. For example, carbon contamination results in significant photons flux losses (practically at the Carbon K edges), and modifications of the horizontal-to-vertical polarization transmission ratio, which degrade with time as the thickness of the carbon layer builds up. This contamination is visible and consists of a gray/black line over the entire photon beam footprint. Addressing the carbon contamination issue, two cleaning processes have been tested quite successfully on two SOLEIL beamlines (in the UV-VUV and soft X-ray ranges), namely in-situ oxygen plasma and in-situ ozone generation via UV lamps. A dedicated group is currently working on the improvement of the cleaning processes, the metrology of the optics before and after cleaning and the study of the carbon coating in order to propose possible strategies to prevent or slow down the contamination process.

Resume. Ce papier presente une etude de miroirs interferentiels multicouches B4C/Mo en vue d’utilisation dans la fabrication des reseaux MultiCouches Alternes (MCA). L’objectif consiste donc a etudier particulierement la formation des couches d’interface entre deux materiaux consecutifs. Les multicouches sont deposees sur des substrats polis de silicium. Elles sont caracterisees par reflectometrie en incidence rasante. Les ajustements numeriques a l’aide d’un modele a trois couches montrent la formation a l’echelle nanometrique d’une couche d’inter-diffusion a l’interface B4C/Mo.