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 ∼5 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.