Visualization of β‐carotene and starch granules in plant cells using CARS and SHG microscopy

Abstract

Information on the content and bioavailability of provitamin A carotenoids, such as β-carotene, in plant foods is of great interest due to the widespread vitamin A deficiency in developing countries. While the amount of β-carotene can readily be quantified with analytical techniques, there is limited information on β-carotene morphology in native plant materials. Here, we introduce nonlinear microscopy for three-dimensional, label-free imaging of carotenoids in fresh and thermally treated plant tissues, providing quantitative information at single-aggregate level and detailed insight into their distribution. Carotenoids in orange-fleshed sweet potato (OFSP), carrot, and mango were visualized by coherent anti-Stokes Raman scattering (CARS) microscopy and, in the case of OFSP, related to the plant-matrix morphology by simultaneous second-harmonic generation (SHG) microscopy of starch granules. Sizes, shapes, densities, and location of different types of carotenoid bodies were quantified. While OFSP and carrot showed heterogeneous rod-shaped bodies with high carotenoid densities indicated by higher CARS signals, the carotenoid-filled lipid droplets in mango appeared as homogeneous low-density aggregates of rounded shape. In addition, β-carotene densities and morphologies in OFSP were studied after thermal processing, showing that the bodies remain intact despite significant changes of the surrounding starch granules.

Visualization of structural changes in cellulosic substrates during enzymatic hydrolysis using multimodal nonlinear microscopy

“…Glucan chains lack inversion symmetry and have previously been shown to give rise to SHG in starch (Brackmann et al 2011;Slepkov et al 2010) and cellulosic substrates of different origin Brown et al 2003;Glas et al 2015;Pohling et al 2014;Slepkov et al 2010;Zimmerley et al 2010). In order to interpret the SHG images of cellulosic substrates, the coherent nature of the SHG signal and the supramolecular structure of cellulose must be taken into account.…”

Raman Spectroscopy of Microbial Pigments

“…Coherent anti-Stokes Raman scattering microscopy (CARS) has been identified as an additional technique for advanced imaging of carotenoids in microalgae and cyanobacteria. In this technique, interfering fluorescence effects are efficiently suppressed (107)(108)(109).…”

In plantaimaging of Δ9-tetrahydrocannabinolic acid inCannabis sativa L.with hyperspectral coherent anti-Stokes Raman scattering microscopy

“…[5][6][7][8][9][10][11] The primary advantages of CARS microscopy over other label-free techniques are its rapid imaging capabilities, which extend up to video rate, 12 and intrinsic confocality, allowing three-dimensional sectioning without descanning beams or the installation of pinholes. We recently introduced a technique for hyperspectral imaging that combines the imaging speed of CARS with the wide tunability of an optical parametric oscillator (OPO).…”