A Method for Preparing Difficult Plant Tissues for Light and Electron Microscopy

Abstract

Although the advent of microwave technologies has both improved and accelerated tissue processing for microscopy, there still remain many limitations in conventional chemical fixation, dehydration, embedding, and sectioning, particularly with regard to plant materials. The Proteaceae, a family of plants widely distributed in the Southern Hemisphere and well adapted to harsh climates and nutrient-poor soils, is a perfect example; the complexity of Proteaceae leaves means that almost no ultrastructural data are available as these are notoriously difficult to both infiltrate and section. Here, a step-by-step protocol is described that allows for the successful preparation of Banksia prionotes (Australian Proteaceae) leaves for both light and transmission electron microscopy. The method, which applies a novel combination of vibratome sectioning, microwave processing and vacuum steps, and the utilization of an ultra low viscosity resin, results in highly reproducible, well-preserved, sectionable material from which very high-quality light and electron micrographs can be obtained. With this, cellular ultrastructure from the level of a leaf through to organelle substructure can be studied. This approach will be widely applicable, both within and outside of the plant sciences, and can be readily adapted to meet specific sample requirements and imaging needs.

Direct fluorescence imaging of lignocellulosic and suberized cell walls in roots and stems

“…Glutaraldehyde and OsO 4 will cross-link and further stabilize subcellular components and have been used in correlative studies by light and electron microscopy ( Kiernan 2000 ; Watanabe et al 2006 ). These three chemicals can be applied sequentially on the same plant material, firstly FAA, then GA to provide fixation of relatively large samples, then followed by fixation with OsO 4 of subsamples for light and electron microscopy ( Kitin et al 2002 , 2009 ; Clode 2015 ). It has to be pointed out that the strong, chemically reactive fixatives are usually toxic if allowed to contact living tissue.…”

“…Microtomes are designed to provide sections with precisely determined and uniform thickness. Vibratomes are helpful for cutting soft plant organs such as leaves, shoots and young roots ( Gunawardena et al 2007 ; Clode 2015 ). Sliding (sledge) microtomes are the most common instrument in anatomical studies of wood or tree bark and are typically employed for making thick sections in the range from 10 to 60 µm of thickness ( Jansen et al 1998 ; Dié et al 2012 ; Delvaux et al 2013 ; Kitin and Funada 2016 ).…”

A First Ultrastructural and Immuno-Labelling Investigation of Western Red Cedar Leaves

“…Table 1 summarizes many fixation and embedding procedures tried to embed WRC leaves completely for morphological investigation. We were limited against possible other techniques, such as microwave-assisted processing (Clode 2015), Tokuyasu’s cryo-sectioning method (1978) and vitrification (high-pressure freezing) followed by freeze substitution (McDonald 2014), as we have access to only a basic Electron Microscope Facility. The alternative techniques require expensive equipment and training beyond our, and many, EM facilities.…”

Systematic evaluation of chromatin immunoprecipitation sequencing to study histone occupancy in dormancy transitions of grapevine buds

“…However, the hair-like structures inside buds create air spaces that could impede penetration of the crosslinking solution. The application of a vacuum cycle procedure was used here to change the pressure around the buds and remove entrapped air, thus allowing more efficient infiltration ( Li et al 2014 b , Clode 2015 ). To test the efficiency of our vacuum infiltration technique, we performed de-crosslinking followed by DNA extraction using the PCI method.…”