Label-free chemically specific imaging in planta with stimulated Raman scattering microscopy.

The growing world population puts ever-increasing demands on the agricultural and agrochemical industries to increase agricultural yields. This can only be achieved by investing in fundamental plant and agrochemical research and in the development of improved analytical tools to support research in these areas. There is currently a lack of analytical tools that provide noninvasive structural and chemical analysis of plant tissues at the cellular scale. Imaging techniques such as coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy provide label-free chemically specific image contrast based on vibrational spectroscopy. Over the past decade, these techniques have been shown to offer clear advantages for a vast range of biomedical research applications. The intrinsic vibrational contrast provides label-free quantitative functional analysis, it does not suffer from photobleaching, and it allows near real-time imaging in 3D with submicrometer spatial resolution. However, due to the susceptibility of current detection schemes to optical absorption and fluorescence from pigments (such as chlorophyll), the plant science and agrochemical research communities have not been able to benefit from these techniques and their application in plant research has remained virtually unexplored. In this paper, we explore the effect of chlorophyll fluorescence and absorption in CARS and SRS microscopy. We show that with the latter it is possible to use phase-sensitive detection to separate the vibrational signal from the (electronic) absorption processes. Finally, we demonstrate the potential of SRS for a range of in planta applications by presenting in situ chemical analysis of plant cell wall components, epicuticular waxes, and the deposition of agrochemical formulations onto the leaf surface.

Single-crystal X-ray diffraction and NMR crystallography of a 1:1 cocrystal of dithianon and pyrimethanil.

A single-crystal X-ray diffraction structure of a 1:1 cocrystal of two fungicides, namely dithianon (DI) and pyrimethanil (PM), is reported [systematic name: 5,10-dioxo-5H,10H-naphtho[2,3-b][1,4]dithiine-2,3-dicarbonitrile-4,6-dimethyl-N-phenylpyrimidin-2-amine (1/1), C14H4N2O2S2·C12H13N2]. Following an NMR crystallography approach, experimental solid-state magic angle spinning (MAS) NMR spectra are presented together with GIPAW (gauge-including projector augmented wave) calculations of NMR chemical shieldings. Specifically, experimental 1H and 13C chemical shifts are determined from two-dimensional 1H-13C MAS NMR correlation spectra recorded with short and longer contact times so as to probe one-bond C-H connectivities and longer-range C...H proximities, whereas H...H proximities are identified in a 1H double-quantum (DQ) MAS NMR spectrum. The performing of separate GIPAW calculations for the full periodic crystal structure and for isolated molecules allows the determination of the change in chemical shift upon going from an isolated molecule to the full crystal structure. For the 1H NMR chemical shifts, changes of 3.6 and 2.0 ppm correspond to intermolecular N-H...O and C-H...O hydrogen bonding, while changes of -2.7 and -1.5 ppm are due to ring current effects associated with C-H...π interactions. Even though there is a close intermolecular S...O distance of 3.10 Å, it is of note that the molecule-to-crystal chemical shifts for the involved sulfur or oxygen nuclei are small.