Visualizing Spatial and Temporal Responses of Plant Cells to the Environment
Lydia-Marie Joubert1* (firstname.lastname@example.org), Michael F. Schmid1, Yue Rui2, Jose Dinneny2, Wah Chiu1, and Peter Dahlberg1
1SLAC National Accelerator Laboratory; and 2Stanford University
This project will develop correlative light and electron microscopy (CLEM) cryo-electron tomography (cryo-ET) tools for high-resolution imaging of fluorescent biosensors in plant cells under different physiological stress conditions.
The plant cell wall is a complex dynamic structure that functions as the primary interface through which interactions between plants and their environment are mediated. To study the nanometer-scale structural changes that correspond to a plant’s response to stress, researchers are developing cryogenic correlative light and electron microscopy methods that use fluorescent biosensors that report on various aspects of a cell’s physiology.
Cryogenic preparation of plant cells has been notoriously challenging due to various morphological and physiological features that are unique to terrestrial plants and dissimilar from both unicellular and multicellular organisms, cell types, and macromolecules for which robotic plunge-freezing techniques were developed. Due to their thick cellulosic cell wall, relatively large size, and water-filled large vacuoles, high-pressure freezing techniques need to be optimized to ensure complete vitrification of the entire plant cell. Using root tips of Arabidopsis seedlings grown on agar and in liquid medium, the team uses cryo-focused ion beam–scanning electron microscopy (cryoFIB-SEM) milling, including Cryo-LiftOut (CLO) techniques, to prepare ultrathin lamellae for cryo-ET data collection. The project additionally uses integrated fluorescence microscopy (iFLM) approaches to do guided milling and target specific fluorescent biosensors for tomographic data collection.
The tools and workflows developed here will be transformative for the field of cryo-ET by demonstrating that fluorescence can be used in cryogenic CLEM experiments to characterize specific aspects of the subcellular chemical environment.
Figure 1. Preparation of root tips for high-pressure freezing using a Leica EM ICE High-Pressure Freezer.
This research was supported by the DOE Office of Science, Biological and Environmental Research (BER) Program grant no. DE-AC02-76SF00515 under FWP 100883.
Some of this work was performed at the Stanford-SLAC CryoET Specimen Preparation Center (SCSC), which is supported by the National Institutes of Health Common Fund’s Transformative High Resolution Cryoelectron Microscopy program (U24GM139166).
Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515.
Dr. Peter Dahlberg is supported by the Department of Energy, Laboratory Directed Research and Development program at SLAC National Accelerator Laboratory, under contract DE-AC02-76SF00515 and as part of the Panofsky Fellowship awarded to Dr. Peter Dahlberg.