Cuticle and subsurface ornamentation of intact plant leaf epidermis under confocal and superresolution microscopy

Abstract

Plant cuticle micromorphology is an invaluable tool in modern ecology and paleoecology. It has expanded our knowledge of systematic relationships among diverse plant groups and can be used to identify fossil plants. Furthermore, fossil plant leaf micromorphology is used for reconstructing past environments, most notably for estimating atmospheric CO concentration. Here we outline a new protocol for imaging plant cuticle for archival and paleoecological applications. Traditionally, both modern reference and fossil samples undergo maceration with subsequent imaging via environmental SEM, widefield fluorescence, or light microscopy. In this paper, we demonstrate the capabilities of alternative preparation and imaging methods using confocal and superresolution microscopy with intact leaf samples. This method produces detailed three-dimensional images of surficial and subsurface structures of the intact leaf. Multiple layers are captured simultaneously, which previously required independent maceration and microtome steps. We compared clearing agents (chloral hydrate, KOH, and Visikol); mounting media (Eukitt and Hoyer's); fluorescent stains (periodic acid Schiff, propidium iodide); and confocal vs. superresolution microscopes. We conclude that Eukitt is the best medium for long-term preservation and imaging. Because of nontoxicity and ease of procurement, Visikol made for the best clearing agent. Staining improves contrast and under most circumstances PAS provided the clearest images. Supperresolution produced higher clarity images than traditional confocal, but the information gained was minimal. This new protocol provides the botanical and paleobotanical community an alternative to traditional techniques. Our proposed workflow has the net benefit of being more efficient than traditional methods, which only capture the surface of the plant epidermis. Microsc. Res. Tech. 81:129-140, 2018. © 2016 Wiley Periodicals, Inc.

Comparison of Sample Preparation Techniques for Inspection of Leaf Epidermises Using Light Microscopy and Scanning Electronic Microscopy

“…Different preparation and imaging methods can affect the quality of leaf sample images (Kawai et al, 2013;Morawetz, 2013;Urban et al, 2018). Research related to methods for observing leaf micro-morphology provides alternative preparation and imaging methods, and improves work efficiency (Schwab and Hülskamp, 2010; Talbot and White, 2013a;Talbot and White, 2013b;Jayakody et al, 2017).…”

“…A comparison of LM, LSCM, and SEM images obtained using different specimen preparation and imaging methods would allow suitable methods to be chosen based on the experimental objective and equipment available, which could save money and improve work efficiency. Although several studies have compared different specimen preparation and imaging methods using an SEM or an 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.…”

Optical coherence tomography versus microscopy for the study of Aloe Vera leaves

“…The main advantage of OCT is its non-invasiveness, which recommends this method for in-vivo analysis of stomata, an important part of a leaf. It is well-known that any environmental changes can have major implications on plant stress [29],29], therefore by measuring and analyzing particular changes in plant structures, one can identify environmental modifications. In this context, the interest in portable non-invasive devices for monitoring leaves becomes compelling.…”

Biophotonic approach for the characterization of initial bitter-rot progression on apple specimens using optical coherence tomography assessments

“…However, these time consuming and destructive experimental procedures affect the biological nature of specimens, and ultimately reduce the experimental accuracy. In fact, most extensively investigated non-destructive plant related disease inspection techniques, such as visual inspection, magnetic resonance imaging (MRI), X-rays, positron emission tomography (PET), confocal microscopy, fluorescence spectroscopy, and near infra-red (NIR) spectroscopy, have been implemented by numerous research groups 9 – 16 . However, low resolution, long acquisition times, and limited depth penetration, are major drawbacks of these methods, which ultimately limit their applications in agriculture.…”