Abstract
The micro-morphology of leaf epidermises is valuable for the study of leaf development and function, as well as the classification of plant species. There have been few studies comparing different preparation and imaging methods for visualizing the leaf epidermis. Here, four specimen preparation methods were used to investigate the leaf epidermis morphology of Arabidopsis, radish, cucumber, wheat, rice, and maize, under an inverted basic light microscope (LM), a laser scanning confocal microscope (LSCM), or a scanning electron microscope (SEM). Optical microscope specimens were obtained using either the direct isolation method or the chloral hydrate-based clearing method. SEM images were obtained using a standard stage for conventional dehydrated samples or a Coolstage for fresh tissue. Different parts of epidermis peels were well focused under the LM. Investigation of samples cleared by chloral hydrate is convenient and autofluorescence of cell walls can be detected in rice. The resolution of images of conventional SEM leaf samples was generally higher than the Coolstage images at the same magnification, whereas local collapse and shrinkage were observed in leaves with high water content when using the conventional method. However, stomatal apparatuses of Arabidopsis, cucumber, radish, and maize deformed and showed poor appearance when using the Coolstage. Moreover, we usually used glutaraldehyde as an SEM fixative when using t-butanol for freeze-drying, though methanol is considered a better fixative in recent studies. In addition, fresh samples were not stable on the Coolstage. Thus, we compared four different t-butanol freeze-drying methods and two Coolstage methods. The dimension and morphology of tissues were compared using the six different methods. The results indicate that methanol fixative obviously reduced shrinkage of SEM samples compared with glutaraldehyde and formaldehyde alcohol acetic acid (FAA) fixatives. The use of methanol and a graded series of steps improved the preservation of samples. Preparing samples with optimal cutting temperature compound and observing at −30°C helped to increase the stability of Coolstage samples. In summary, our results provide an overview of the shortcomings and merits of four different methods, and might provide some information about choosing an optimal method for visualizing epidermal morphology.
Highlights
The leaf is the primary site of organic compound production and gas exchange
Methods for Observing Leaf Epidermis have compared different specimen preparation and imaging methods using an scanning electronic microscope (SEM) or an light microscope (LM) (Uwins et al, 1993; Choi et al, 2016; Urban et al, 2018), there remains a lack of studies focused on choosing a suitable method and microscope for visualizing epidermis morphology
Plant epidermis samples were obtained by peeling (Arabidopsis, radish, and cucumber) or scraping and observed under an inverted LM equipped with a color camera (Figures 1 and 2 and Figures S1D–F)
Summary
The leaf is the primary site of organic compound production and gas exchange. Observing the micro-structure of the leaf epidermis is of important theoretical and applied value. One solution is to separate the epidermis from the mesophyll tissue during sample preparation using methods such as direct isolation (by peeling or scraping), peeling after boiling in maceration agents, for example, chromic acid/nitric acid (Stockey et al, 1995; Shah et al, 2018). Another method for visualizing the epidermis is to prepare transparent leaf tissues using clearing agents such as KOH, NaOH, hypochlorite, and chloral hydrate (Morawetz, 2013; Shah et al, 2018; Vovides et al, 2018). There are other methods that have been developed to investigate leaf micro-morphology, including making impressions of the leaf epidermis with clear fingernail polish (Waisel et al, 1969; Gitz and Baker, 2009)
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