Development of advanced imaging approaches for enabling plant biology research

Co-sponsored by the Department of Plant Pathology and Environmental Microbiology

Abstract:
Our work seeks to develop and optimize multiscale volume electron microscopy (vEM) and multiplex correlative approaches that enable new insights on the structure-function relationship and sub-cellular distribution of targeted molecules in plant research. However, unique challenges are faced with plant specimen due to their waxy cuticles, cell walls and air spaces which impede fixation and downstream sample preparation. Additionally, these same features induce optical aberrations for photon imaging and result in poorly conductive samples for volume electron microscopy, severely limiting accessibility for high-resolution interrogation in intact plants. To address these limitations, the application and optimization of multiscale microscopy for plants can serve as an invaluable approach to more easily identify, relocate and image target structures and maintain context within bulk tissues.

Improvements in cryo-workflows and heavy metal staining strategies for 3D vEM interrogation of plant samples is rapidly evolving. Resin-based electron microscopy preparations with improved heavy metal staining are highly amenable for vEM multiscale correlative workflows using x-ray microscopy (XRM) and along with nanoscale 3D reconstructions of whole plant cells. Cryo-workflows, the gold-standard in cellular preservation, allow imaging of bulk frozen-hydrated plant specimen using vEM without chemical pre-treatment and can be combined with cryo-fluorescence to identify critical cell targets. In all instances, artificial intelligence is routinely leveraged for both XRM and vEM datasets for improved image quality and/or segmentation.

Multiplex microscopy simultaneously or sequentially visualizes an extended array of biomolecular probes within cells, thus providing high-dimensional physical mapping of numerous cellular phenotypes and components over heterogeneous tissues. While multiplex fluorescent probe labeling on cryo-sections and thin acrylic sections is well established in mammalian tissues, adoption in plants has been very limited. Strategies for the application of correlative multiplex microscopy using confocal and/or super-resolution imaging followed by back-scatter scanning electron microscopy plant samples will be described.

Ultimately, the adoption and adaptation of these disparate multiscale and multiplex correlative approaches to address the specific challenges of plants is a “work in progress” but have shown tremendous potential in our ongoing efforts. Further development and dissemination of robust protocols will be instrumental in supporting the broader plant microscopy community.

About the Speaker:
Dr. Kirk Czymmek received his doctorate in the Department of Botany and Plant Pathology at Michigan State University in 1993 followed by a post-doctoral position at the DuPont Company in CR&D Plant Molecular Genetics group. Subsequently, he worked with Noran Instruments as an applications scientist before joining the University of Delaware (UD). At UD, Dr. Czymmek actively worked for 15 years and established the UD Bio-Imaging Center at the Delaware Biotechnology Institute, 2001. He served as its Director as an Associate Professor in the Department of Biological Sciences with research in fungal cell biology and plant pathogenesis. In 2012, he joined ZEISS to build a world-class application, demonstration and training center for the ZEISS microscopy portfolio for North America which subsequently led to his role as Vice President Global ZEISS Microscopy Customer Centers and oversight of 8 customer centers world-wide. In 2019, Dr. Czymmek joined the Donald Danforth Plant Science Center as a Principal Investigator and Director the Advanced Bioimaging Laboratory to apply and develop advanced microscopy tools in plant science dedicated to producing more nutritious food and improving the environment. In 2022 he launched the start-up Peptyde Bio to develop antimicrobial peptides for crop protection as a co-founder and Chief Technology Officer and sold to Invaio in December 2023. With over 30 years of advanced microscopy experience, he has expertise in most forms of light, x-ray and electron microscopy, including super-resolution microscopy, cryo-techniques and correlative microscopy. Dr. Czymmek has over 120-refereed publications focused on developing and applying cutting-edge microscopy tools for imaging cells, tissues and biomaterials.

Contact

Seogchan Kang
sxk55@psu.edu