Label-free Chemically Specific Imaging in Planta with Stimulated Raman Scattering Microscopy

Abstract

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 non-invasive 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 labelfree 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 allows near realtime imaging in 3D with submicron 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.

In Vivo Chemical and Structural Analysis of Plant Cuticular Waxes Using Stimulated Raman Scattering Microscopy

“…Because SRS is detected at the same wavelength as the excitation fields, it is not affected by fluorescent emission and, as recently demonstrated by Mansfield et al (2013), may also be used in the presence of highly pigmented samples such as plant tissues.…”

“…We have recently shown that it is possible use phasesensitive detection to separate the vibrational SRS signal from the electronic absorption processes (Mansfield et al, 2013). In this investigation, we used SRS imaging to investigate both the structure and chemical composition of plant cuticular waxes, simultaneously and in vivo.…”

“…Stimulated Raman gain was measured in preference to stimulated Raman loss, as it allowed the chemically specific Raman signal to be separated from any additional signal due to two photon absorption (Ye et al, 2009) or photothermal lensing (Lu et al, 2010;Moger et al, 2012) by phase-sensitive lock-in detection (Mansfield et al, 2013).…”

“…Fresh, excised plant leaves were mounted in perfluorodecalin (Littlejohn et al, 2010) prior to imaging, which has been shown not to interfere with Raman-based imaging in plants (Mansfield et al, 2013;Littlejohn et al, 2014).…”

Contrast in coherent Raman scattering microscopy

“…Experimental requirements for the nonlinear optical imaging of plants are significantly different than for animal samples [115,116]. Many plant cells contain high concentrations of efficient light-absorbing molecules, such as chlorophyll, that limit the penetration of light into the sample and substantially reduce the optical damage threshold compared to transparent animal cells.…”