Three-Dimensional Imaging of Plant Cell Wall Deconstruction Using Fluorescence Confocal Microscopy

Abstract

Lignocellulosic biomass (LB) is recalcitrant to enzymatic hydrolysis due to its compact and complex cell wall structure. To identify the parameters behind LB recalcitrance, experimental data over hydrolysis time must be collected. Here, we describe a novel method to collect time-lapse images during cell wall deconstruction by enzymatic hydrolysis. The protocol includes instructions for sample preparation, layout of a custom designed incubation chamber and instructions for confocal time lapse acquisition. The protocol sets out a detailed plan where cross-sections of untreated and pretreated poplar samples are mounted in a sealed frame containing a buffer and an enzymatic cocktail. The sealed frame is then placed into an incubator to maintain the sample at a constant temperature of 50 °C, which is optimal for enzymatic reaction while avoiding enzymatic cocktail evaporation. Using lignin natural autofluorescence, confocal z-stacks of untreated and pretreated samples were acquired at regular time intervals during enzymatic hydrolysis for 24 h. Acquisition parameters were optimized to compromise between image resolution and reduced photo-bleaching. The acquired image might then be processed by further development of algorithms to extract precise quantitative information on cell wall deconstruction. This protocol is an important first step towards elucidating the underlying parameters of LB recalcitrance by allowing the acquisition of high-quality images of LB hydrolysis for extracting quantitative data on LB deconstruction.

Plant phenotyping relevance

ポプラ細胞壁の分解状態を時系列の共焦点3D画像で取得するプロトコル自体が中心であり、植物組織状態の定量的表現型抽出を可能にするため。

Code and data availability

The paper's authors state that the scripts for computing photobleaching signal loss and image registration/analysis are publicly available in the FARE Laboratory GitLab repository, with an explicit URL matching an allowed URL.