Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

The overall goal of this procedure is to determine the location and structure of calcification event by performing a combination of optical and electron microscopy methods. This is accomplished by monitoring the living larval tube worm during metamorphosis, the life stage responsible for calcification that can be detected using fluorescent indicators sensitive to intracellular pH and calcium signals. Intracellular pH imaging allows us to study the concentration of proton into and out of the cytoplasm in the process of calcification.

To begin, intracellular pH imaging in the marine tube worm larvae, the competent swimming larvae are stained with the intracellular pH indicator dye, SNARF-1 AM.The larvae are then transferred to a dish of IBMX containing seawater with a thin glass observing window and placed on a fluorescent microscope, where they are hit with light at 488 nanometers in wavelength, which is absorbed by the dye. 580 nanometer and 640 nanometer emissions from the dye are collected. The values of intracellular pH can be calculated from the ratios of these values.