4D Imaging of Brown Algal Cells

Area of Science:

• Marine Biology
• Cell Biology
• Microscopy

Background:

• In vivo 3D imaging of brown algae is hindered by light-diffracting pigments (fucoxanthin, chlorophyll) and high-salinity seawater.
• Conventional fluorescence microscopy limits 4D (x, y, z, t) imaging of brown algal developmental processes.
• Existing fluorochromes like calcofluor white (CFW) have limitations in depth penetration and potential for aggregation in seawater.

Purpose of the Study:

• To develop and detail a protocol for robust, multicolor in vivo 4D imaging of brown algal cell shape.
• To introduce and validate a new orange-emitting fluorochrome, styryl benzoindoleninium sulfonate (SBIS), for plasma membrane labeling.
• To enable long-term, non-toxic live imaging of brown algal development using confocal and light sheet microscopy.

Main Methods:

• Developed a step-by-step protocol for labeling brown algal tissues with SBIS.
• Optimized mounting techniques for 3D confocal and light sheet time-lapse microscopy.
• Established imaging parameters to minimize toxicity and enhance signal-to-noise ratio for brown algal cell visualization.

Main Results:

• SBIS effectively labels the plasma membrane of brown algal cells, enabling multicolor imaging.
• The protocol allows visualization of brown algal cells up to 25 μm thickness and for up to 7 days.
• Successful 4D imaging was demonstrated in various brown algae species, including *Ectocarpus* sp., *Sphacelaria rigidula*, and *Saccharina latissima*.

Conclusions:

• The SBIS protocol provides a robust method for 4D visualization of brown algal cell shape during growth.
• This technique overcomes previous imaging limitations, offering broad applications for cellular-level studies in brown algae.
• The optimized protocol supports live in vivo imaging without interfering with brown algal development.