Visualizing endomembranes with confocal microscopy in protoplasts and cells of tobacco

The distribution and redistribution of the endoplasmic reticulum (ER) was studied with Confocal Laser Scanning Microscopy (CLSM) during the regeneration of tobacco protoplasts. In freshly isolated protoplasts the ER is cisternal in appearance, forming aggregates of plate-like structures clustered around the chloroplasts. During the cell's development, the ER profiles first lose the clustered appearance, then gradually tubular forms develop. Finally mixed population of some cisternal and tubular shapes and a lot of discrete spherical or cylindrical vesicles is spread through the cell's cytoplasm. The probe 3,3'-dihexyloxacarbocyanine iodide (DiOC₆(3)) proved to be a very efficient tool, allowing observations with a resolution of 0.2 μm.