The potential of Raman microscopy and Raman imaging in plant research

Abstract

To gain a better understanding on structure, chemical composition and properties of plant cells, tissues and organs several microscopic, chemical and physical methods have been applied during the last years. However, a knowledge gap exists about the location, quantity and structural arrangement of molecules in the native sample or what happens on the molecular level when samples are chemically or mechanically treated or how they respond to mechanical stress. These questions need to be answered to optimise utilization of plants in food industry and pharmacy and to understand structure-function relationships of plant cells to learn from natures unique. Advances in combining microscopy with Raman spectroscopy have tackled this problem in a non-invasive way and provide chemical and structural informationin situwithout any staining or complicated sample preparation. In this review the different Raman techniques (e.g. near infrared Fourier Transform Raman spectroscopy (NIR-FT), resonance Raman spectroscopy, surface-enhanced Raman spectroscopy) are briefly described before approaches in plant science are summarised. Investigations on structural cell wall components, valuable plant substances, metabolites and inorganic substances are included with emphasis on Raman imaging. The introduction of the NIR-FT-Raman technique led to many applications on green plant material by eliminating the problem of sample fluorescence. For mapping and imaging of whole plant organs (seeds, fruits, leaves) the lateral resolution (~10μm) of the NIR-FT technique is adequate, whereas for investigations on the lower hierarchical level of cells and cell walls the high resolution gained with a visible laser based system is needed. Examples on high resolution Raman imaging are given on wood cells, showing that changes in chemistry and orientation can be followed within and between different cell wall layers having dimensions smaller than 1 μm. In addition imaging the distribution of amorphous silica is shown on horsetail tissue, including an area scan from a cross section as well as a depth profiling within a silica rich knob of the outer stem wall.

Vibrational spectroscopic mapping and imaging of tissues and cells

“…Conversely, apart from the advantage of being able to use FT techniques with 1064-nm excitation, the lack of fluorescence, which often accompanies visible irradiation with biological samples and swamps the Raman spectrum, is an additional advantage. These advantages combined often outweigh the disadvantages and have resulted in the extensive use of FT-Raman studies on biological samples (Gierlinger and Schwanninger 2007;Moreira et al 2008;Movasaghi et al 2007;Parker 1994).…”

“…Because of the rapid expansion of biological applications of vibrational spectroscopic mapping and imaging techniques, it is not possible to provide an extensive review of recent research within the space limitations of this article, but an overview of the diversity of applications in medicine and biology, including medical diagnostics, studies of physiological and diseases processes and treatments of diseases and plant biology, can be found in a number of recent reviews (Bailo and Deckert 2008;Bhargava 2007;Boskey and Mendelsohn 2005;Chan et al 2008;Gierlinger and Schwanninger 2007;Levin and Bhargava 2005;Moreira et al 2008;Movasaghi et al 2007;Petibois and Guidi 2008;Petter et al 2009;Srinivasan and Bhargava 2007;Swain and Stevens 2007). As outlined in these reviews and the current review, the increase in the speed of acquisition, sensitivity, discrimination and spatial resolution of the emerging techniques of vibrational spectroscopic microscopies has greatly expanded the horizons of what is possible in terms of an explosion of new knowledge that is being generated.…”

Comparison of a compatible and an incompatible pepper-tobamovirus interaction by biochemical and non-invasive techniques: Chlorophyll a fluorescence, isothermal calorimetry and FT-Raman spectroscopy

“…An other non-destructive technique, the Fourier-transformation Raman (FT-Raman) spectroscopy has also been applied to study the chemical composition and properties of plant tissues (Gierlinger and Schwanninger, 2007), including plants exposed to abiotic and biotic stress effects (Taddei et al, 2002;Skoczowski and Troc, 2013). This method was used for in situ analysis of primary and secondary metabolites in living plant tissues in two different ways: by pointmeasurements or by two dimensional Raman mapping, which characterizes the distribution of these compounds (Skoczowski and Troc, 2013).…”

Quantifying Bio-Engineering: The Importance of Biophysics in Biofuel Research

“…All major cell wall biopolymers are both IR and Raman active. Nowadays both techniques are widely used in plant cell wall research (Dokken et al, 2005;Gierlinger and Schwanninger, 2007). Unlike most of the current techniques for cell wall compositional analysis, such as wet chemistry assays, chromatography methods, mass spectrometry etc., which are destructive and involve breakdown of the cell wall components or extensive chemical treatment of the plant cell walls, IR and Raman spectroscopy can characterize cell wall components in their native form with minimal requirement for sample preparation.…”

“…This system is often coupled to a confocal microscope equipped with objectives with high numerical apertures to achieve high spatial resolution (Gierlinger and Schwanninger, 2007;Smith and Dent, 2005). For example, a Raman spectrometer with a 514.5 nm laser was used to study the concentration of lignocellulosics in the cell corner middle lamella of both birch and spruce (Tirumalai et al, 1996).…”