Algae to angiosperms: Autofluorescence for rapid visualization of plant anatomy among diverse taxa
Timothy J. Pegg, Daniel K. Gladish, Robert L. Baker
Abstract
PREMISE: Fluorescence microscopy is an effective tool for viewing plant internal anatomy. However, using fluorescent antibodies or labels hinders throughput. We present a minimal protocol that takes advantage of inherent autofluorescence and aldehyde-induced fluorescence in plant cellular and subcellular structures to markedly increase throughput in cellular and ultrastructural visualization. METHODS AND RESULTS: Twelve species distributed across the plant phylogeny were each subjected to five fixative treatments: 1% paraformaldehyde and 2% glutaraldehyde, 2% paraformaldehyde, 2% glutaraldehyde, formalin-acid-alcohol (FAA), and 70% ethanol. Samples were prepared by embedding and mechanically sectioning or via whole mount. A confocal laser scanning system was used to collect micrographs. We evaluated and compared fixative influence on sample structural preservation and tissue autofluorescence. CONCLUSIONS: Formaldehyde fixation of Viridiplantae taxa samples generates useful structural data while requiring no additional histological staining or clearing. In addition, a fluorescence-capable microscope is the only specialized equipment required for image acquisition. The minimal protocol developed in this experiment enables high-throughput sample processing by eliminating the need for multi-day preparations.