Abstract

A novel microspectrophotometer is described, which simultaneously resolves cell absorption into two mutually orthogonal components allowing the determination of linear dichroism as a function of wavelength in the range of 325–695 nm. This instrument uses a single plane-polarized light beam and a small general-purpose digital computer, and is equipped with a photo-flash apparatus for rapid photolysis. Following visual pigment bleaching, it can detect changes occurring on a time scale of seconds in the orientation and spectral character of chromophores in isolated cells. The spectral scanning is performed in either single or multiple sweeps which may be unidrectional or bidirectional. The scanning rate is set to 500 nm/s. Spectral resolution is 5 nm. Its signal and data processing are discussed. Its performance is illustrated on subcellular organelles of retinal photoreceptors from turtle and frog. Rhodopsin and its photoproducts are shown to lend dichroism to frog rod outer segments. Metarhodopsin II, when formed, is transversely dichroic as rhodopsin. The late products (retinol, retainal oxime, etc.) show axial dichroism. The corrected specific optical density (transverse component) of frog rod outer segments (in hydroxylamine) is found to be 0.0182±0.002/μm. The average absorption spectrum is presented for in situ rhodopsin.

© 1974 Optical Society of America

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