Monday, April 1, 2013

Confocal microscopy

This is a confocal projection of a chiton trochophore larva preserved with formaldehyde and stained with fluorescent phalloidin. Confocal microscopy allows one to collect a series of images of thin (e.g. 1 micron or less) optical sections from a small, fluorescently labeled specimen (like this ~200 micron long larva), excluding the out-of-focus light. One can then create a z-projection of the entire stack, as I did here.

Phalloidin binds to filamentous actin, so when it is fluorescently labeled it allows visualization of cell outlines and muscles, among other structures. The feature of interest on this image is what appears to be a transverse band of small cells about two thirds of the way to the anterior end (up) of the larva.  This is the prototroch. The prototroch is a transverse ciliary band anterior to the mouth, and is the defining feature of a trochophore larva. In chiton trochophores it is the main locomotory organ. 

The small, grid-shaped blocks that catch one's eye are not actually individual cells within the prototroch but rather a pattern of actin fibers near the surface of the prototroch cells. The cells of the prototroch are much larger.  One can visualize the outlines of the prototroch cells by removing the top few slices of the image stack (second picture). 

The image at left is a 10-micron-thick optical slice (in mid-sagittal plane) of a specimen, that was dehydrated through an isopropanol series and cleared in a mixture of benzyl benzoate and benzyl alcohol (Murray Clear) to visualize internal structures. Larval anterior is up, and ventral is to the left (marked by the position of the mouth). The bowling pin shape in the center is the body-wall musculature composed of longitudinal and circular fibers and located directly beneath the epidermis. These muscles act antagonistically to control the larva's locomotion and shape. The prototroch cells are visible here as the dark regions on either side of the larva, near the narrow neck of the "bowling pin." The mouth opens immediately posterior to the prototroch on the ventral side (left). The opening and the lumen of the foregut are also highlighted with phalloidin. Phalloidin labeling in combination with confocal microscopy is an excellent tool for studies of external and internal morphology of small embryos and larvae.

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