SBIS, a new orange fluorescent vital probe for the 4D imaging of brown algal cells

Living cells of brown algae are difficult to observe in 3D due to the presence of pigments such as fucoxanthin and chlorophyll that diffract light. Furthermore, they grow slowly in seawater. To gain insight into the shape and size of brown alga cells in 3D during the development of these multicellular photosynthetic organisms, we designed a fluorescence probe that efficiently and selectively labels the plasma membrane. Styryl benzoIndoleninium sulfonate (SBIS) is a bright orange fluorogenic probe that is soluble and virtually non-emissive in seawater; it accumulates in the plasma membrane of brown algal cells, where it activates upon binding. SBIS enabled the 3D observation of cells of different shapes and sizes in several brown alga species, over a period of up to seven days and in a medium mimicking natural growth conditions. More importantly, SBIS makes it possible to observe cells at thicknesses of up to 20 µm, in contrast to Calcofluor White. These properties resulted in the visualization of the entire contour of the cells in the Z-axis of uniseriate filaments of Ectocarpus, polystichous filaments of Sphacelaria and cellular monolayered lamina of Saccharina . Altogether, SBIS now makes the study of embryogenesis and early development possible in 4D in these brown algae.