Kirk James Czymmek

Cryogenic volume electron microscopy (cryo-vEM) is a rapidly developing nanoscale imaging approach that enables three-dimensional datasets of vitrified cells and tissues at intermediate resolutions without treatment with chemical fixatives or metal contrasting agents. Our goal was to evaluate the suitability of cryo-vEM for whole cells of photosynthetic organisms such as cyanobacteria. Two species of Anabaena , a filamentous cyanobacterium, were captured on poly-L-lysine-coated grids, vitrified by plunge-freezing into liquid ethane, and mapped by secondary electron and integrated fluorescence light microscopy to identify target cells. Cells of interest were coated with platinum, trenched with xenon, and 3D volumes collected by alternating argon milling and through-lens detector (TLD) imaging on a Thermo Scientific Hydra plasma focused ion beam scanning electron microscope. Volumes were segmented via 2.5D U-Net models, then quantified in Dragonfly 3D World. Block-face images revealed the cell wall, thylakoids, phycobilisomes, carboxysomes, cyanophycin and polyphosphate granules. Whole-cell 3D rendering allowed quantification of carboxysomes, cyanophycin granules, and measurement of thylakoid thickness and spacing in A. inaequalis and Anabaena sp. UTEX 2576. Thus, cryo-vEM provided interpretable whole-cell ultrastructure of cyanobacteria without chemical pre-treatment, and its compatibility with cryo-fluorescence and in situ cryo-ET enables contextual multi-scale perspectives from whole cells to molecular structures.

Plasmodesmata (PD) provide plant viruses a direct route for cell-to-cell spread. Viral infection increases intercellular trafficking via PD, termed gating, but the mechanism involved remains unclear. Here, using a combination of high-resolution volume electron microscopy and live cell imaging, we demonstrate that diverse viruses increased the number of PD in infected leaves. This increase was triggered by viral movement proteins and the induction of PD formation depended on the presence of the virus and the localization of movement proteins to PD. Further, Group I Remorins, known inhibitors of virus infection, are negative regulators of PD formation and inhibit virus or movement protein-induced changes to PD density. Our results lead to a model in which viral gating of PD may result from increased de novo PD formation.

As part of two open international core facility surveys focused on microscopy and flow cytometry, we obtained an in‐depth snapshot of their state‐of‐play. Here we focus on microscopy cores across ‘the Americas’, ‘Asia Pacific’ and ‘Europe and Africa’ with responses from 241 facilities. We compared instrument types and numbers, staffing, user fees, funding, etc. This revealed both remarkable similarities and notable differences. For example, the staff/instrument ratio in Europe and Africa was smaller compared to other regions World View and Asia Pacific had a larger proportion of electron microscopes. Importantly, we want to emphasise that the anonymised raw data is made available for anyone to do their own further mining and analysis, including cross‐correlations and constrained groupings. This survey will serve as an essential baseline for future longitudinal studies following trends across all metrics as our facilities evolve.

One of the greatest challenges when generating large and/or high-resolution three-dimensional (3D) volume electron microscopy (vEM) datasets is long acquisition times. We developed a method that leverages Artificial intelligence algorithms to increase acquisition throughput for 3D datasets by creating an AI-derived mask for the targeted region of interest, referred to as Adaptive Scanning. This allowed for specific structures to be imaged at high resolution, with the surrounding area captured at lower resolution, without artificially generating or enhancing any raw data. We demonstrate that this dynamic-resolution scanning approach significantly reduced acquisition time depending on the number of pixels of interest and was sample agnostic, being compatible with a diverse array of organisms and tissues, including brains, parasites, cultured cells, and plants. This multiresolution strategy has the potential to enhance focused ion beam scanning electron microscopy and other forms of vEM, by increasing time savings by up to twofold or more, enabling routine generation of multiple and/or larger vEM datasets more efficiently, cost-effectively, and allowing more data collection to increase statistical power for comparative studies.

Cell walls underpin the mechanics of cell growth, intercellular signalling, and defence against pathogenic organisms. β-(1,3)-glucans (also known as callose) are polysaccharides found in plants, fungi, and some bacterial species. In developing plant organs, callose accumulates around intercellular channels (plasmodesmata) controlling cell-to-cell communication. We developed monoclonal antibodies for the detection of β-(1,3)-glucans and using these identified distinct populations of callose differing in size and secondary structure. Callose sub-populations were in proximal but not overlapping cell wall microdomains surrounding plasmodesmata. Moreover, labelling with an antibody that detects larger more complex β-(1,3)-glucans structures (named LM-BDG2) was absent in Arabidopsis meristems implying distinct spatial and functional microenvironments. Biochemical and studies with mutants suggest that the accumulation of LM-BDG2-labelled callose structures is influenced by interactions with xyloglucans (another plant glycan regulating cell wall properties). This work extends current understanding and demonstrates structural heterogeneity in plant β-(1,3)-glucans. It supports interactions between glycans with roles in the regulation of cell wall properties and functions.

Frogeye Leaf Spot disease, caused by a fungus Cercospora sojina, is a serious disease affecting soybean production. Its control is challenged by resistance to Quinone outside Inhibitor (QoI) fungicides, but plant antifungal peptides with multi-faceted modes of action (MoA) offer a promising alternative. Here, we showed that chickpea nodule-specific cysteine-rich peptide, NCR13_Peptide Folding Variant1 (NCR13_PFV1), exhibited antifungal activity against QoI-sensitive and -resistant field isolates of C. sojina at nanomolar concentrations representing the first antifungal NCR peptide reported effective against C. sojina. Spray-application of this peptide showed no phytotoxicity and effectively protected soybean against FLS. When combined with the QoI fungicide azoxystrobin, NCR13_PFV1 provided additive control of FLS. NCR13_PFV1 disrupted plasma membrane, induced reactive oxygen species (ROS) production, and was rapidly internalized, localizing in the nucleus. It bound to fungal ribosomal RNA and inhibited protein translation. RNA-seq studies revealed the upregulation of several genes encoding heme binding proteins in peptide-challenged C. sojina. Notably, iron supplementation in the growth medium reduced the peptide-induced ROS and antifungal activity, revealing the importance of iron homeostasis in protection or recovery of C. sojina from oxidative stress. Overall, NCR13_PFV1 with its multi-faceted MoA holds potential as a bio-fungicide for FLS control, complementing QoI fungicides and overcoming resistance in C. sojina.

Expansion microscopy is an imaging method that overcomes the resolution barriers of traditional diffraction-limited light microscopes via isotropic physical expansion of biological cells, tissues, and organisms. This technique enhances the ability to visualize proteins, nucleic acids, and other biomolecules at nanoscale resolutions. While this approach has predominantly been applied to biomedical applications, workflows for plant systems have emerged. Here we review the development of expansion microscopy methods for the plant kingdom. These advances range from isolated organelles, single cells, and unicellular organisms to whole organs, embryos, and reproductive tissues. We highlight the unique advantages and findings from individual techniques and provide a perspective of potential directions that expansion microscopy can be applied to answer important plant research questions.

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.

One of the greatest challenges when generating large and/or high-resolution three-dimensional (3D) volume electron microscopy (vEM) datasets is long acquisition times. We developed a method that leverages Artificial Intelligence (AI) algorithms to increase acquisition throughput for 3D datasets by creating an AI-derived mask for the targeted region of interest, referred to as Adaptive Scanning. This allowed for specific structures to be imaged at high resolution, with the surrounding area captured at lower resolution, without artificially generating or enhancing any raw data. We demonstrate that this dynamic-resolution scanning approach significantly reduced volume acquisition time across a diverse array of organisms and tissues, including brains, parasites, cultured cells, and plants. This multi-resolution strategy has the potential to enhance Focused Ion Beam Scanning Electron Microscopy and other forms of vEM, by increasing time savings by up to 2-fold or more, enabling routine generation of multiple and/or larger vEM datasets, more efficiently and cost-effectively, allowing more data collection for increase statistical power for comparative studies.