Optical and X-Ray Multimodal-Hybrid Microscope Systems for Imaging of Plant-Pathogen Interactions
Authors:
Soichi Wakatsuki1,2* ([email protected], PI), Rose Knight1,2, Dara Dowlashahi1,2, Adam Bowman2,4, David Nana Agyeman-Budu1,2, James Safranek1, Johanna Nelson Weker1, Jung-Gun Kim2, Christopher Takacs1, Jacob Summers2, Mary Beth Mudgett2, Federica Brandizzi3, Mark Kasevich2
Institutions:
1SLAC National Accelerator Laboratory; 2Stanford University; 3Michigan State University; 4Salk Institute for Biological Studies
Abstract
Plant-pathogen interactions are complex and dynamic phenomena, relevant to fundamental, environmental and bioenergy biology. Imaging plays important roles in understanding the interactions at the atomic, molecular, subcellular, cellular, to tissue and whole plant scales. Each bioimaging method has its intrinsic limitations in spatial and/or temporal resolution, field of view and depth, and sensitivity. Fluorescence-based optical microscopes have huge advantages in monitoring dynamics of cellular and subcellular events using wide spectral ranges with super-resolution, light-sheet 3D, and wide-field imaging, but cannot go beyond 10 or 20 nanometers spatial resolution. X-ray imaging and tomography can penetrate deeper than light, and with high brilliance synchrotron sources one can reach 10 nm spatial resolution, but with a high risk of sample damage from radiation dose. Cryogenic electron tomography (cryo-ET) can offer tremendous insight into the subcellular organization of organelles and macromolecules down to several nm resolution, but information gained is largely static. Large efforts have gone into correlative imaging but, to date, there is no easy way to correlate more than two modalities directly. Correlating these different modalities in an unequivocal manner will substantially advance understanding of multiscale complex biological phenomena.
This talk presents updates on the development of the next generation correlative X-ray, light, and electron tomography systems (see figure). In particular, the team has modularized electro-optic fluorescence lifetime imaging microscopy (EO-FLIM) (Bowman et al. 2019; Bowman and Kasevich 2021) for use in correlative microscopies, in the deep ultraviolet, or at synchrotron beamlines, preparing for a first implementation alongside transmission X-ray microscopy (TXM), beamline 6-2c, with correlative X-ray/vis optics at SLAC and Stanford Synchrotron Radiation Lightsource. The team will utilize both the micro- and nano-computed tomography (CT) imaging capability of beamline 6-2 to gain hierarchical insights into the dynamics of fungal-pathogen interactions. Towards this end, the team has successfully collected preliminary tomography data using a laboratory-based micro-CT X-ray source on plant root and leaf samples. The team has also established a standing EO-FLIM microscope at the synchrotron for further uncorrelated data collection and to explore extremes of resonant drive frequencies.
Finally, the team presents advances towards fluorescent protein- and nanocrystal-containing cages as molecular tracers for X-ray imaging/microscopy and EO-FLIM and fiducial markers for cryo-ET, including proof-of-concept synchrotron TXM images of leaf samples bombarded with 400 nm nanogold particles. The presentation discusses further plans for the initial application examining fungal-plant pathogen interactions via chitin binding domains at the plant cell surface and subsequent applications in an ongoing study of extracellular vesicles.
Image
Electro-Optic Fluorescence Lifetime Imaging Microscopy. (A, B) Micro-computed tomography of Arabidopsis thaliana root sample at the cellular length scale. (C) Proposed lock-in synchronization between X-ray photoluminescence, X-ray microscopy, and FLIM to give both (D) EO-FLIM and (E) X-ray images. [Courtesy SLAC National Accelerator Laboratory]
References
Bowman, A. J., and M. A. Kasevich. 2021. “Resonant Electro-Optic Imaging for Microscopy at Nanosecond Resolution,” ACS Nano 15(10), 16043–54. DOI:10.1021/ acsnano.1c04470.
Bowman, A. J., et al. 2019. “Electro-Optic Imaging Enables Efficient Wide-Field Fluorescence Lifetime Microscopy,” Nature Communications 10(1) DOI:10.1038/s41467-019-12535-5.
Funding Information
This research is supported by the DOE Office of Science, through the Biomolecular Characterization and Imaging Sciences program, BER program, under BCIS FWP 100878.