Kombination af Raman Imaging og Multivariate Analysis til Visualisering af Lignin, Cellulose og Hemicellulose i Plant Cell Wall

Summary

Denne protokol har til formål at fremlægge en generel metode til visualisering af lignin, cellulose og hemicellulose i plantecellevægge ved anvendelse af Raman-billeddannelse og multivariatanalyse.

Abstract

Anvendelsen af ​​Raman-billeddannelse til plantebiomasse er stigende, fordi den kan tilbyde rumlige og sammensatte oplysninger om vandige opløsninger. Analysen kræver normalt ikke omfattende prøvepræparation; Strukturelle og kemiske oplysninger kan opnås uden mærkning. Imidlertid indeholder hvert Raman-billede tusindvis af spektre; Dette rejser vanskeligheder ved udtrækning af skjulte oplysninger, især for komponenter med lignende kemiske strukturer. Dette arbejde introducerer en multivariabel analyse for at løse dette problem. Protokollen etablerer en generel metode til visualisering af hovedkomponenterne, herunder lignin, cellulose og hemicellulose inden i plantecellevæggen. I denne protokol beskrives procedurer til prøveudarbejdelse, spektraloptagelse og databehandling. Det er stærkt afhængig af operatør færdigheder ved prøve forberedelse og data analyse. Ved at anvende denne fremgangsmåde kan en Raman-undersøgelse udføres af en ikke-specialist bruger til at erhverve higH-kvalitetsdata og meningsfulde resultater til analyse af plantecellevægge.

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

1. Prøveforberedelse Skær en lille vævsblok (ca. 3 mm x 3 mm x 5 mm) fra planteprøven ( f.eks. En poppelstamme). Dyp vævet i kogende deioniseret vand i 30 minutter. Overfør straks det til deioniseret vand ved stuetemperatur (RT) i 30 minutter. Gentag dette trin, indtil vævet synker til bunden af ​​beholderen, hvilket indikerer at luften i vævet er fjernet, og at vævet er blødgjort. Bemærk: For de prøver, der synker til bunden forud for dette trin, gentages denne cyklu…

Representative Results

Figur 1 viser et overblik over et typisk mikro-Raman-system til Raman-billeddannelsen af ​​en plantecellevæg. Som et eksempel har de oprindelige Ramanspektre af poppel ( Populus nigra L.) betydelige baseline drift og pigge ( figur 2a ). Efter at have udført den automatiske forbehandlingsteknik for Raman-billeddatasæt (APRI) fjernes disse to spektrale forureninger med succes ( figur 2b</strong…

Discussion

Planten cellevæg er en sammensætning, der er organiseret i flere lag, herunder cellehjørne (CC), sekundærvæg (SW, med S1, S2 og S3 lag), og sammensatte midter lameller (CML, midter lamell plus tilstødende primær Væg), hvilket gør det vanskeligt at opnå en flad overflade under prøvefremstilling. Således skal planteprøver, især græs, som har en mere kompliceret struktur end træ, ofte størkne for at muliggøre fint snitning. PEG er en ideel hård matrix til skæring og Raman undersøgelse, da den er opløs…

Materials

Microtome	Thermo Scientific	Microm HM430
Confocal Raman microscope	Horiba Jobin Yvon	Xplora
Oven	Shanghai ZHICHENG	ZXFD-A5040