An Overview of Cryo-Scanning Electron Microscopy Techniques for Plant Imaging

Abstract

Many research questions require the study of plant morphology, in particular cells and tissues, as close to their native context as possible and without physical deformations from some preparatory chemical reagents or sample drying. Cryo-scanning electron microscopy (cryoSEM) involves rapid freezing and maintenance of the sample at an ultra-low temperature for detailed surface imaging by a scanning electron beam. The data are useful for exploring tissue/cell morphogenesis, plus an additional cryofracture/cryoplaning/milling step gives information on air and water spaces as well as subcellular ultrastructure. This review gives an overview from sample preparation through to imaging and a detailed account of how this has been applied across diverse areas of plant research. Future directions and improvements to the technique are discussed.

An R2R3 MYB transcription factor PsFLP regulates the symmetric division of guard mother cells during stomatal development in Pisum sativum

“…If there are still ice crystals on the surface, sublimate again to remove ice until all ice crystals on the surface of the sample are eliminated. After deicing, the samples were first sprayed with a vacuum ion sputtering instrument for 90 s and then observed and imaged four at a voltage of 5 kV under high vacuum mode under an electron microscope (Wightman, 2022).…”

Visualisation of microalgal lipid bodies through electron microscopy

“…This results in cells that are in a near-native state, with fewer artefacts compared to a chemical fixation. 29,30 Several cryo-immobilisation methods exist, such as plunge freezing, spray freezing, contact freezing or high-pressure freezing. 29 Plunge freezing in cryogenic liquids is a fast and commonly used method.…”

“…29,30 Several cryo-immobilisation methods exist, such as plunge freezing, spray freezing, contact freezing or high-pressure freezing. 29 Plunge freezing in cryogenic liquids is a fast and commonly used method. As the Leidenfrost effect might slow down the freezing rate, using liquid nitrogen slush is a better choice because it is colder than liquid nitrogen.…”

Plasma-Derived Fibrin Hydrogels Containing Graphene Oxide for Infections Treatment

“…Despite other complementary techniques are widely used to characterize fiber-based hydrogels, such as by supercritical point drying process, 41,50 both cryo-SEM and cryo-FIB-SEM are highly recommendable novel techniques to characterize biological materials, since both of them allow the hydrogel visualization in its native state, without any previous sample treatment that could modify the network structure and/or composition. 51 In the case cryo-FIB-SEM, 52 the technique combines a SEM with a focused ion beam (FIB), by which the electron beam images the cryo-sample surface while the ion beam mills into the surface to expose the interior of the sample generating a trench, and allowing the study of the biological material in 3D (Figure S5 in the Supporting Information) and in a fully hydrated state due to the sample vitrification process. Three different samples were tested: the pregel of PLASMA_0, as well as PLASMA_0 and PLASMA_0.1 after 20 min of polymerization.…”

“…The samples were also characterized by cryo-SEM and cryo-FIB-SEM in order to investigate the structure of the hydrogels. Despite other complementary techniques are widely used to characterize fiber-based hydrogels, such as by supercritical point drying process, , both cryo-SEM and cryo-FIB-SEM are highly recommendable novel techniques to characterize biological materials, since both of them allow the hydrogel visualization in its native state, without any previous sample treatment that could modify the network structure and/or composition …”