Polarizing microscopy of eyespot of Chlamydomonas: In situ observation of its location, orientation, and multiplication

The eyespot in the cell of Chlamydomonas reinhardtii has been found to appear as a bright spot under the cross polar setting of a polarizing microscope. This was confirmed by isolating the eyespot from a homogenate of wall-deficient mutant cw-15, and by observing it under a polarizing microscope. Thus, the eyespot was proved to be a strongly birefringent body. Next, gametes (mt⁺ and mt⁻) of 137c strain were prepared by cultivating it in a low-nitrogen (NH₄Cl) medium. Here, every cell shows only one (and never more than one) birefringent spot. The birefringent eyespot was located always near the surface on the “equator,” that is, at the farthest point from the “meridional” cell-axis that is defined as the bisector of the two flagella projected out from the cell surface. It was shown, in addition, that the optic axis of this birefringent eyespot is oriented in the cell always along the parallel direction of the cell axis defined above. Thus, the polarizing microscopy has been shown to provide a powerful method for in vivo, in situ pursuit of the eyespot of Chlamydomonas. © 1999 John Wiley & Sons, Inc. Biospectroscopy 5: 93–100, 1999