Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens

Katharine R. Minker, Meredith L. Biedrzycki, Abhishek Kolagunda, Stephen Rhein, Fabiano J. Perina, Samuel S. Jacobs, Michael Moore, Tiffany M. Jamann, Qin Yang, Rebecca Nelson, Peter Balint-Kurti, Chandra Kambhamettu, Randall J. Wisser, Jeffrey L. Caplan

Research output: Contribution to journalArticle

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.

Original languageEnglish (US)
Pages (from-to)141-152
Number of pages12
JournalMicroscopy research and technique
Volume81
Issue number2
DOIs
StatePublished - Feb 1 2018

Keywords

  • confocal microscopy
  • disease resistance
  • image analysis
  • plant–pathogen interactions
  • tissue clearing

ASJC Scopus subject areas

  • Anatomy
  • Histology
  • Instrumentation
  • Medical Laboratory Technology

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  • Cite this

    Minker, K. R., Biedrzycki, M. L., Kolagunda, A., Rhein, S., Perina, F. J., Jacobs, S. S., Moore, M., Jamann, T. M., Yang, Q., Nelson, R., Balint-Kurti, P., Kambhamettu, C., Wisser, R. J., & Caplan, J. L. (2018). Semiautomated confocal imaging of fungal pathogenesis on plants: Microscopic analysis of macroscopic specimens. Microscopy research and technique, 81(2), 141-152. https://doi.org/10.1002/jemt.22709