Background

The study of cell shapes and sizes in 3D during embryogenesis and growth is a widely studied topic in metazoans, plants, and brown algae. Brown algae (Phaeophyceae) are photosynthetic organisms and one of the five groups of eukaryotes that have acquired complex multicellularity and evolved independently of red algae, green plants (including green algae and land plants), animals, and fungi. In multicellular organisms, 3D growth is considered an intricate morphogenetic process. In metazoans, cellular migration commonly drives the establishment of growth axes in multicellular eukaryotes growing in 3D. However, brown algal cells, like plant cells, are surrounded by a semi-rigid cell wall that cements them together. Consequently, the orientation of 3D growth relies on changes in the cell division planes at specific locations. The abundant natural carotenoid and chlorophyll a pigments present in the thylakoid membranes of brown algal chloroplasts [1] absorb blue-green light (excitation wavelength, 480–500 nm) and emit red light (emission wavelength, 646–655 nm for carotenoid and 677–680 nm for chlorophyll a [2]); this characteristic makes in vivo imaging challenging. Moreover, despite the emergence of genome editing tools in brown algae, which have enabled a knockout to be achieved in Ectocarpus sp. [3], knock-ins are not yet feasible. Therefore, along with the development of microscopy techniques enabling time-lapse studies of these organisms in 3D, research aiming at developing fluorochromes labeling specific compartments of brown algal cells is essential. Several fluorescent dyes have been used to label cellular structures in brown algal tissues, but each presents limitations for long-term 4D imaging. Calcofluor white (CFW), a UV-absorbing and blue-emitting fluorochrome, is capable of forming hydrogen bonds with β (1→4) and β (1→3) polysaccharides, thus binding mainly to the chitin and cellulose present in the cell wall of brown algal cells [4,5]. However, the use of CFW reaches a limit when imaging living organisms in 3D over a long period of time, as UV increases the generation of reactive oxygen species that are toxic to living organisms. Furthermore, blue light is less penetrating into the living tissues than longer wavelengths. Fluorochromes labeling the plasma membrane, like FM1-43 [N-(3-Triethylammoniumpropyl)-4-(4-(Dibutylamino) Styryl) Pyridinium Dibromide] and FM4-64 [dibromure de N-(3-triethylammoniumpropyl)-4-(6-(4-(diethylamino) phenyl) hexatrienyl) pyridinium] are internalized by endocytosis within minutes after labeling Ectocarpus sp., S. rigidula, and S. latissima cells. As a result, they are not suitable for a 4D study of brown algae embryogenesis and growth. The aim of this study is to propose a protocol for visualizing brown algal cells in 4D by optimizing mounting for confocal and light sheet microscopy, adjusting acquisition parameters to minimize phototoxicity, and carefully selecting a fluorochrome that allows clear visualization of the plasma membrane.