Combinazione di Raman Imaging e analisi multivariata per la visualizzazione di lignina, cellulosa e emicellulosa nella parete cellulare delle piante
Summary
Questo protocollo mira a presentare un metodo generale per la visualizzazione della lignina, della cellulosa e dell'emicellulosa nelle pareti delle cellule vegetali utilizzando l'analisi Raman e l'analisi multivariata.
Abstract
L'applicazione di Raman imaging alla biomassa vegetale è in aumento in quanto può offrire informazioni spaziali e compositive sulle soluzioni acquose. L'analisi di solito non richiede un'ampia preparazione dei campioni; Informazioni strutturali e chimiche possono essere ottenute senza etichettatura. Tuttavia, ogni immagine Raman contiene migliaia di spettri; Ciò solleva difficoltà quando estrae informazioni nascoste, soprattutto per i componenti con strutture chimiche simili. Questo lavoro introduce un'analisi multivariata per affrontare questo problema. Il protocollo stabilisce un metodo generale per visualizzare i componenti principali, tra cui la lignina, la cellulosa e l'emicellulosa all'interno della parete cellulare delle piante. In questo protocollo sono descritte le procedure per la preparazione del campione, l'acquisizione spettrale e l'elaborazione dei dati. È altamente dipendente dall'abilità dell'operatore durante la preparazione del campione e l'analisi dei dati. Utilizzando questo approccio, un'indagine di Raman può essere eseguita da un utente non specializzato per ottenere higI dati di qualità h e risultati significativi per l'analisi delle pareti cellulari delle piante.
Introduction
Plant biomass is the most abundant renewable resource on Earth; is mainly composed of lignin, cellulose, and hemicellulose; and is considered an attractive source of bioenergy and bio-based chemicals1. Unfortunately, it can resist degradation and confer hydrolytic stability or structural robustness to the plant cell wall. Such resistance is attributable to the accessible surface area, biomass particle size, degree of polymerization, cellulose crystallinity, and protective lignin2. A comprehensive understanding of the structural and chemical nature of the plant cell wall is thus significant from the viewpoint of plant biology and chemistry, as well as from that of commercial utilization. Commonly used wet chemistry analyses, such as chromatography, mass spectrometry, and nuclear magnetic resonance spectroscopy, only provide average compositional data of the measured sample. Furthermore, these methods are invasive and destroy the original structure of the plant tissue3.
The Raman imaging technique is a powerful tool for the nondestructive visualization of spatially resolved chemical information4. It uses a laser light to cause inelastic scattering with a photon and relies on changes in polarizability arising from the molecular vibrations. In this case, water causes weak Raman scattering, which makes this approach suitable for in situ investigations of biological samples5. The application of the Raman imaging technique to the plant cell wall can elucidate the structure and composition of plant cell walls in their native state, with the resolution on the scale of the single cell and even of the cell wall layers6. A typical Raman imaging analysis of a plant cell wall generally consists of three steps: 1) sample preparation, 2) spectral acquisition, and 3) data processing.
Although one of the major advantages of Raman imaging is the ability to achieve label-free and non-destructive spectra with minimal sample preparation, physical sample sectioning is still necessary to expose the surface of interest. This process should be performed carefully to obtain a flat surface, since the technique depends on maintaining optical focus7. Spectral acquisition requires a balance between image quality and extensive acquisition times8. Data processing aims to effectively extract the chemical information from the image data, especially for the components with similar chemical structures, such as cellulose and hemicellulose. Due to the strong spectral overlap, the exact spectra are difficult to discern. In this case, multivariate analysis is a straightforward approach to effectively uncover the hiding structural and chemical information9. This work presents a general protocol describing the use of Raman imaging to visualize the main components in plant cell walls, including lignin, cellulose, and hemicellulose.
Protocol
Representative Results
Discussion
La parete cellulare è un composito organizzato in diversi strati, tra cui l'angolo di cella (CC), la parete secondaria (SW, con gli strati S1, S2 e S3) e la lamella centrale composta (CML, lamella centrale e principale adiacente Parete) che rende difficile ottenere una superficie piana durante la preparazione del campione. Pertanto, i campioni di piante, in particolare l'erba, che hanno una struttura più complessa del legno, spesso devono essere solidificati per consentire una sezione sottile. PEG è una matri…
Disclosures
The authors have nothing to disclose.
Acknowledgements
Ringraziamo il Ministero della Scienza e della Tecnologia della Cina (2016YDF0600803) per il sostegno finanziario.
Materials
| Microtome | Thermo Scientific | Microm HM430 | |
| Confocal Raman microscope | Horiba Jobin Yvon | Xplora | |
| Oven | Shanghai ZHICHENG | ZXFD-A5040 |
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