Techniques for Imaging the Endoplasm and Vacuole of Characean Algae
Thesis DisciplineCellular and Molecular Biology
Degree GrantorUniversity of Canterbury
Degree NameMaster of Science
The extremely large internodes of the Characean algae have long served as a model system for biologists for investigations of membrane electrophysiology, the cell wall and the cytoskeleton, and the knowledge gleaned from these studies has facilitated subsequent discoveries in other systems, particularly higher plants. Despite this, the cytoplasmic organization of the streaming endoplasm and vacuole in the internodes remain poorly understood. Investigations of cytoplasmic architecture are typically conducted using a mixture of confocal laser scanning microscopy, expression of fluorescent proteins, and membrane-permeant fluorescent dyes. As Characean algae have not been successfully transformed, this study has used confocal microscopy with fluorescent dyes to image live cells. However, this encounters two notable problems, the light-absorbing chloroplast layer that encases all but the cortical regions of the cell, and the fast streaming rates of the endoplasm which distort imaging. Using confocal microscope settings optimized for high speed imaging, two complementary techniques have been used to image the interior of the cells. High intensity irradiation for 5 minutes with a 405 nm violet-laser generated windows in the cortical layer through which the endoplasm and vacuole could be imaged, while the vacuole and endoplasm could also be imaged in the structurally-related rhizoid cells. With these techniques, vivid image sequences of cytoplasmic architecture and dynamics within the endoplasm and vacuole have been achieved with a range of fluorescent dyes, notably neutral red and acridine orange, the cell marker Blue CMAC which loads into the central vacuole, the DNA stain Syto-13, and the tonoplast specific dye MDY-64. These results demonstrate that the streaming endoplasm and vacuole are considerably more structurally complex than previous investigations have described, and that the central vacuole is heterogeneous.