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Cuticle and subsurface ornamentation of intact plant leaf epidermis under confocal and superresolution microscopy
Author(s) -
Urban Michael A.,
Barclay Richard S.,
Sivaguru Mayandi,
Punyasena Surangi W.
Publication year - 2018
Publication title -
microscopy research and technique
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.536
H-Index - 118
eISSN - 1097-0029
pISSN - 1059-910X
DOI - 10.1002/jemt.22667
Subject(s) - confocal , cuticle (hair) , biology , confocal microscopy , microscopy , botany , optical sectioning , pathology , anatomy , optics , microbiology and biotechnology , physics , medicine
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 2 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.