MICROSCOPIC LASER SPECTROSCOPY OF CHICK EMBRYO LENSES

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One of the key factors in cell differentiation and development is the intracellular synthesis of macromolecules specific to the differentiated cells. The chick embryo lens is widely used as a model system in the study of cell differentiation. Its morphology and cytoplasmic protein composition are simple and it is accessible at all stages of development. Considerable effort has been focused on the protein chemistry of the lens using molecular genetic techniques in relation to cataract. We have recently developed a technique of microscopic laser light scattering spectroscopy that allows observation of the Brownian motion of macromolecules within the cytoplasm of a single, live cell. From the amplitude and relaxation time of the fluctuations of light scattered from the macromolecules, it is possible to determine their intracellular concentration and size distribution. With this non-invasive technique, we have succeeded in determining the time course of intracellular accumulation of Delta-crystallin within the chick embryo lens during development. The technique is sensitive and quantitative for the study of the state of cytoplasmic proteins as was demonstrated in a study of hemoglobin aggregation inside human normal and sickle red blood cells. Based on our preliminary data we propose t study the in vivo development of the chick embryo lens, in vitro differentiation of retinal pigmented epithelium into lens cells upon clonal cell culture. Using the technique, the intracellular composition of crystallin proteins will be determined during the differentiation processes. Comparative studies using biochemical techniques will also be carried out. The technique will also be used to study the aggregation process of crystallins associated with osmotic swelling of the lens cells. The studies as proposed will lead to quantitatively better understanding of the development and transdifferentiation of the eye, and shed light on the physical basis for cytoplasmic abnormalities.