A Simple Chamber for Long-term Confocal Imaging of Root and Hypocotyl Development

Charlotte Kirchhelle,Ian Moore

doi:10.3791/55331

Abstract

Several aspects of plant development, such as lateral root morphogenesis, occur on time spans of several days. To study underlying cellular and subcellular processes, high resolution time-lapse microscopy strategies that preserve physiological conditions are required. Plant tissues must have adequate nutrient and water supply with sustained gaseous exchange but, when submerged and immobilized under a coverslip, they are particularly susceptible to anoxia. One strategy that has been successfully employed is the use of a perfusion system to maintain a constant supply of oxygen and nutrients. However, such arrangements can be complicated, cumbersome, and require specialized equipment. Presented here is an alternative strategy for a simple imaging system using perfluorodecalin as an immersion medium. This system is easy to set up, requires minimal equipment, and is easily mounted on a microscope stage, allowing several imaging chambers to be set up and imaged in parallel. In this system, lateral root growth rates are indistinguishable from growth rates under standard conditions on agar plates for the first two days, and lateral root growth continues at reduced rates for at least another day. Plant tissues are supplied with nutrients via an agar slab that can be used also to administer a range of pharmacological compounds. The system was established to monitor lateral root development but is readily adaptable to image other plant organs such as hypocotyls and primary roots.

Highlights

To study cellular and subcellular processes that underlie plant development, there is an increasing demand for high-resolution long-term timelapse imaging strategies
Lateral root lengths of plants in imaging chambers were measured at hourly intervals (Figure 2A, left, Movie 1, n = 23) and growth rates were compared to those of lateral roots grown on standard Petri plates containing the same agar-solidified medium (Figure 2A, right, Movie 2, n = 23)
While growth rates generally increased with the length of the root, growth rates still varied substantially between roots of similar length but the variance was similar in imaging chambers and on plates

Summary

Introduction

To study cellular and subcellular processes that underlie plant development, there is an increasing demand for high-resolution long-term timelapse imaging strategies. Systems like the RootChip[2] and the RootArray[3] have been designed for time lapse imaging of developing roots and involve germinating seeds in a custom-build multi-specimen device. These arrangements ensure minimal mechanical perturbation and are designed for the parallel analysis of multiple seedlings, but are not optimized for imaging of subcellular structures. This agar slab gently presses the root against the coverslip, fixing its relative position in the imaging chamber and allowing the use of high-resolution water immersion lenses. The imaging chambers were developed and characterized to study lateral root development but are adaptable to imaging other seedling organs such as primary root tips and hypocotyls

Creating the Chamber

Creating the Agar-solidified Slab of Growth Medium

Finishing the Chamber Setup

Representative Results

Discussion