Optical monitoring of freeze-trapped reaction intermediates in protein crystals: a microspectrophotometer for cryogenic protein crystallography

A microspectrophotometer for cryogenic protein crystallography is described. It is capable of measuring visible absorption spectra (350-800 nm) of a single crystal during X-ray data collection. The microspectrophotometer is designed to minimize the level of stray light by using a double monochromator and an optical microscope equipped with two field diaphragms and a pinhole. In this system, a thick crystal with an optical density of similar to5 is measurable. In order to demonstrate the performance of the system, the absorption spectra of the unphotolyzed state and the primary photoreaction intermediate of bacteriorhodopsin in the P622 crystal have been measured under a flow of cold nitrogen gas at 100 K.