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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.

Ultraviolet (UV) synchrotron radiation circular dichroism (SRCD) spectroscopy has made an important contribution to the determination and understanding of the structure of bio-molecules. In this paper, we report an innovative approach that we term time-resolved SRCD (tr-SRCD), which overcomes the limitations of current broadband UV SRCD setups. This technique allows accessing ultrafast time scales (down to nanoseconds), previously measurable only by other methods, such as infrared (IR), nuclear magnetic resonance (NMR), fluorescence and absorbance spectroscopies, and small angle X-ray scattering (SAXS). The tr-SRCD setup takes advantage of the natural polarization of the synchrotron radiation emitted by a bending magnet to record broadband UV CD faster than any current SRCD setup, improving the acquisition speed from 10 mHz to 130 Hz and the accessible temporal resolution by several orders of magnitude. We illustrate the new approach by following the isomer concentration changes of an azopeptide after a photoisomerization. This breakthrough in SRCD spectroscopy opens up a wide range of potential applications to the detailed characterization of biological processes, such as protein folding and protein-ligand binding. [ABSTRACT FROM AUTHOR]

DISCO, a novel low-energy beamline covering the spectrum range from the VUV to the visible, has received its first photons at the French synchrotron SOLEIL. In this article the DISCO design and concept of three experimental stations serving research communities in biology and chemistry are described. Emphasis has been put on high flux generation and preservation of polarization at variable energy resolutions. The three experiments include a completely new approach for microscopy and atmospheric pressure experiments as well as a 'classical' synchrotron radiation circular dichroism station. Preliminary tests of the optical design and technical concept have been made. Theoretical predictions of the beam have been compared with the first images produced by the first photons originating from the large-aperture bending-magnet source. Results are also reported concerning the cold finger used to absorb hard X-ray radiation in the central part of the synchrotron beam and to avoid heavy thermal load on the following optics. Wavelength selection using monochromators with different gratings for each experimental set-up as well as beam propagation and conditioning throughout the optical system are detailed. First photons comply very well with the theoretical calculations. [ABSTRACT FROM AUTHOR]