Comprehensive Guide Sets New Standards for Plant Fluorescence Imaging

A team of expert scientists led by Kirk Czymmek, PhD, director of the Advanced Bioimaging Laboratory at the Donald Danforth Plant Science Center and Heather E. McFarlane, assistant professor at the University of Toronto and collaborators from the Danforth Center, University of Leeds (UK), University of Massachusetts (Amherst), University of California - Davis, University of Naples Federico II, University of Minnesota and Université de Montréal, have authored a comprehensive guide to elevate the quality, transparency, and reproducibility of fluorescence microscopy in plant research. The guide was recently published in the journal Plant Cell, "Best Practices in Plant Fluorescence Imaging and Reporting: A Primer"

Microscopy is a fundamental approach used for plant cell and developmental biology as well as an essential tool for mechanistic studies in plant research. However, setting up a new microscopy-based experiment can be challenging, especially for beginner users, when implementing new imaging workflows or when working in an imaging facility where staff may not have extensive experience with plant samples. The basic principles of optics, chemistry, imaging, and data handling are shared among all cell types. However, unique challenges are faced when imaging plant specimens due to their waxy cuticles, strong/broad spectrum autofluorescence, recalcitrant cell walls, and air spaces that impede fixation or live imaging, impacting sample preparation and image quality. 

Our goal was to compile a resource that goes beyond methodological know-how-to elevate competency, transparency and reproducibility in plant imaging across the community. This guide offers plant-specific advice and examples for microscope users at all stages of fluorescence microscopy workflows, from experimental design through sample preparation, image acquisition, processing, and analyses, to image display and methods reporting in manuscripts." 

Kirk Czymmek, PhD, Director, Advanced Bioimaging Laboratory, Donald Danforth Plant Science Center 

Topics included:

• Addressing Plant-Specific Imaging Challenges:Fluorescence microscopy in plants faces unique obstacles-waxy cuticles, strong autofluorescence, rigid cell walls, and air-filled tissues can degrade image clarity and impede live imaging.
• End-to-End Workflow Guidance:The primer offers thorough recommendations covering experimental design, probe selection, sample prep, imaging modalities, image processing, data handling, quantification, and final reporting-creating a unified workflow framework for all researchers, especially newcomers.
• Focus on Reproducibility & Reporting Standards:The authors outline vital metadata disclosure, standards for image display, and guidelines for documenting parameters to support reproducibility and enable future meta-analyses.
• Illustrative Examples & Tools Provided:The primer includes practical illustrations-such as comparing widefield, confocal, and super-resolution imaging-and shows protocols to reduce photobleaching, improve signal-to-noise, and manage image compression artifacts, with visual examples demonstrating best vs. suboptimal practices.

The resource, created as part of the National Science Foundation-funded Plant Cell Atlas initiative led by Seung Yon (Sue) Rhee, Director of Michigan State University's Plant Resilience Institute, provides a baseline standard for plant imaging protocols, enhancing data integrity and enabling researchers across labs to harmonize methodologies. As plant research increasingly relies on complex imaging data, clear best practices are essential for cumulative progress and cross-study comparisons.

"One of the central goals of the Plant Cell Atlas is to empower the plant science community with resources that raise the bar for rigor, transparency, and collaboration. This primer embodies that mission by providing clear, plant-specific guidance for fluorescence microscopy, enabling researchers worldwide to generate high-quality, reproducible data that will accelerate discovery," said Rhee.