Confocal Microscopy

Auricularia

Confocal microscopy is highly specialized optical imaging technique that can create 3D models of larvae. It does this by stacking cross sectional images, obtained by shooting lasers through the specimen. The lasers detect fluorescent markers of different wavelengths.
Three fluorescent markers were used: DAPI, phalloidin, and primary antibody. DAPI, in blue, binds directly to DNA, highlighting nuclei. Phalloidin, in green, binds F-actin and highlights muscle. Primary antibody made in a mouse, binds tubulin. A 546 nm fluorescent secondary antibody, anti-mouse made in goat, bound the primary antibody and is shown in magenta. The tubulin marks the ciliary band in the auricularia, and the nervous system in the pentacula.

There is a clear concentration of nuclei in the stomach, hydropore, and ciliary band. This indicates that cells forming the epidermis are much larger, and most likely less specialized. The magenta highlights the complex shape of the ciliary band. Surprisingly, the only muscles were the esophageal muscles. This implies that all mobile movement of auricularia is from beating cilia.

Unlike auricularia, individual nuclei are generally evenly distributed throughout the pentacula. The magenta shows a rich network of neurons that concentrate at the ends of the buckle tentacles and tube feet, which are used to sense. The buckle tentacles and tube feet are also rich in muscle. This is also not surprising, as the pentacula use both these organs to move around the sea floor. The phalloidin also beautifully highlights the longitudinal body retractor muscles that surround the ambulacra, and contractile ring muscles along the inner walls of the cucumber.

Pentacula