Observing ER Dynamics over Long Timescales Using Light Sheet Fluorescence Microscopy.

ABSTRACT

The recent significant progress in developmental bio-imaging of live multicellular organisms has been greatly facilitated by the development of light sheet fluorescence microscopy (LSFM). Both commercial and custom LSFM systems offer the best means for long-term rapid data collection over a wide field of view at single-cell resolution. This is thanks to the low light exposure required for imaging and consequent limited photodamage to the biological sample, and the development of custom holders and mounting techniques that allow for specimens to be imaged in near-normal physiological conditions. This method has been successfully applied to plant cell biology and is currently seen as one of the most efficient techniques for 3D time-lapse imaging for quantitative studies. LSFM allows one to capture and quantify dynamic processes across various levels, from plant subcellular compartments to whole cells, tissues, and entire plant organs. Here we present a method to carry out LSFM on Arabidopsis leaves expressing fluorescent markers targeted to the ER. We will focus on a protocol to mount the sample, test the phototoxicity of the LSFM system, set up a LSFM experiment, and monitor the dynamics of the ER during heat shock.