title: Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens.
abstract: The study of phenotypic variation in plant pathogenesis provides fundamental information about the nature of disease resistance. Cellular mechanisms that alter pathogenesis can be elucidated with confocal microscopy; however, systematic phenotyping platforms-from sample processing to image analysis-to investigate this do not exist. We have developed a platform for 3D phenotyping of cellular features underlying variation in disease development by fluorescence-specific resolution of host and pathogen interactions across time (4D). A confocal microscopy phenotyping platform compatible with different maize-fungal pathosystems (fungi: Setosphaeria turcica, Cochliobolus heterostrophus, and Cercospora zeae-maydis) was developed. Protocols and techniques were standardized for sample fixation, optical clearing, species-specific combinatorial fluorescence staining, multisample imaging, and image processing for investigation at the macroscale. The sample preparation methods presented here overcome challenges to fluorescence imaging such as specimen thickness and topography as well as physiological characteristics of the samples such as tissue autofluorescence and presence of cuticle. The resulting imaging techniques provide interesting qualitative and quantitative information not possible with conventional light or electron 2D imaging. Microsc. Res. Tech., 81:141-152, 2018. © 2016 Wiley Periodicals, Inc.
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, 19716.
authors.forename: Katharine R
authors.initials: KR
authors.lastname: Minker
authors.affiliation: Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware, 19716.
authors.forename: Meredith L
authors.initials: ML
authors.lastname: Biedrzycki
authors.affiliation: Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware, 19716.|Department of Computer and Information Sciences, University of Delaware, Newark, Delaware, 19716.
authors.forename: Abhishek
authors.initials: A
authors.lastname: Kolagunda
authors.affiliation: Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware, 19716.|Department of Computer and Information Sciences, University of Delaware, Newark, Delaware, 19716.
authors.forename: Stephen
authors.initials: S
authors.lastname: Rhein
authors.affiliation: Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware, 19716.
authors.forename: Fabiano J
authors.initials: FJ
authors.lastname: Perina
authors.affiliation: Department of Bio-Imaging Center, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711.
authors.forename: Samuel S
authors.initials: SS
authors.lastname: Jacobs
authors.affiliation: Department of Bio-Imaging Center, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711.
authors.forename: Michael
authors.initials: M
authors.lastname: Moore
authors.affiliation: Department of Crop Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois, 61801.
authors.forename: Tiffany M
authors.initials: TM
authors.lastname: Jamann
authors.affiliation: Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina, 27695.
authors.forename: Qin
authors.initials: Q
authors.lastname: Yang
authors.affiliation: School of Integrative Plant Science, Cornell University, Ithaca, New York, 14853.
authors.forename: Rebecca
authors.initials: R
authors.lastname: Nelson
authors.affiliation: Department of Plant Pathology, North Carolina State University, Raleigh, North Carolina, 27695.|USDA-ARS, Plant Science Research Unit, Raleigh, North Carolina, 27695.
authors.forename: Peter
authors.initials: P
authors.lastname: Balint-Kurti
authors.affiliation: Department of Computer and Information Sciences, University of Delaware, Newark, Delaware, 19716.
authors.forename: Chandra
authors.initials: C
authors.lastname: Kambhamettu
authors.affiliation: Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware, 19716.
authors.forename: Randall J
authors.initials: RJ
authors.lastname: Wisser
authors.affiliation: Department of Biological Sciences, University of Delaware, Newark, Delaware, 19716.|Department of Bio-Imaging Center, Delaware Biotechnology Institute, University of Delaware, Newark, Delaware, 19711.
authors.forename: Jeffrey L
authors.initials: JL
authors.lastname: Caplan
mesh_terms: D001331:Automation; D005658:Fungi / Q000472:pathogenicity*; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D018613:Microscopy, Confocal / Q000379:methods*; D061848:Optical Imaging / Q000379:methods; D010935:Plant Diseases / Q000382:microbiology; D013048:Specimen Handling / Q000379:methods; D013194:Staining and Labeling / Q000379:methods; D003313:Zea mays / Q000382:microbiology*
keywords: confocal microscopy; disease resistance; image analysis; plant-pathogen interactions; tissue clearing