Best practices in plant fluorescence imaging and reporting: A primer

Microscopy is a fundamental approach 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. As expert plant microscopists, we share our collective experience on best practices to improve the quality of published microscopy results and promote transparency, reproducibility, and data reuse for meta-analyses. We offer 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. We also present standards for methods reporting that will be valuable to all users and offer tools to improve reproducibility and data sharing.