Environmental scanning electron microscopy (ESEM)—a versatile tool in studying plants

Abstract

Environmental scanning electron microscopy (ESEM) enables the investigation of hydrated and uncoated plant samples and the in situ observation of dynamic processes. Water vapor in the microscope chamber takes part in secondary electron detection and charge prevention. Two ESEM modes are available and offer a broad spectrum of applications. The environmental or wet mode prevents sample dehydration by the combination of sample cooling (5°C) and a vapor pressure of 4-6 Torr. In the low vacuum mode, the maximum chamber pressure is limited to 1 Torr (corresponding to about 5% relative humidity in the chamber) and allows the simultaneous use of a backscattered electron detector for imaging material contrast. A selection of characteristic plant samples and various applications are presented as a guide to ESEM for plant scientists. Leaf surfaces, trichomes, epicuticular waxes, and inorganic surface layers represent samples being comparatively resistant to dehydration, whereas callus cells and stigmatic tissue are examples for dehydration- and beam-sensitive samples. The potential of investigating dynamic processes in situ is demonstrated by studying anther opening, by tensile testing of leaves, and by performing hydration/dehydration experiments by changing the vapor pressure. Additionally, automated block-face imaging and serial sectioning using in situ ultramicrotomy is presented. The strengths and weaknesses of ESEM are discussed and it is shown that ESEM is a versatile tool in plant science.

Morphological changes induced by advanced glycation endproducts in osteoblastic cells: Effects of co-incubation with alendronate

“…One important advance in microscopy has been the introduction of the environmental scanning electron microscope (E-SEM) that can operate at very low vacuum and allows the observation of fresh samples without the need for any previous treatment (Stabentheiner et al, 2010). The development of this instrumentation means that whole new classes of materials can now be observed in their natural state (Uroukov and Patton, 2008;Osma et al, 2011).…”

In Situ Experiments in the Scanning Electron Microscope Chamber

“…Several works are specifically dedicated to in situ experimentation. Stabentheiner et al (2010) state that "one unrivaled possibility of ESEM is the in situ investigation of dynamic processes that are impossible to access with CSEM where samples have to be fixed and processed". These authors have studied the anther opening that is a highly dynamic process involving several tissue layers and controlled tissue desiccation.…”

“…This field is one of the most interesting uses of the ESEM that offers fantastic opportunities for matter properties characterisation. Even if numerous recent articles and reviews are dedicated to in situ experimentation in the VP/ESEM (Donald, 2003 ;Mendez-Vilas et al, 2008 ;Stokes, 2008 ;Stabentheiner et al, 2010 ;Gianola et al, 2011 ;Torres & Ramirez, 2011), no one describes all the possibilities of this technique. The present chapter will provide a large -and as exhaustive as possible -overview of the possibilities offered by the new SEM and ESEM generation in terms of "in situ experiments" focussing specifically on the more recent results (2000)(2001)(2002)(2003)(2004)(2005)(2006)(2007)(2008)(2009)(2010)(2011).…”

“…Thiel & Donald (1998) and Stabentheiner et al (2010) describe the deformation of plants (carrots and leaves respectively) during room temperature tensile tests performed in the ESEM chamber. Similar tests are also reported with food (Stokes & Donald, 2000) and they are regularly performed on polymers (Poelt et al, 2010;Lin et al, 2011), composites (Schoßig et al, 2011) and metals (Boehlert et al, 2006;Gorkaya et al, 2007).…”

“…with a large depth of field and with grey level contrasts. In-SEM experimentation can be extended to a wide range of applications, corresponding to very different materials (plants (Stabentheiner et al, 2010), food (Thiel et al, 2002 ;James, 2009), paper (Manero et al, 1998), soft matter, polymers, metals, ceramics, solids, liquids…) or problems (plant behaviour, chemical reactivity, properties characterization, sintering, grain growth, corrosion…). In the literature, the main part of the data reported has been acquired using an environmental scanning electron microscope.…”