Optical monitoring of protein crystals in time-resolved x-ray experiments: Microspectrophotometer design and performance

We describe a single crystal microspectrophotometer for optical monitoring of protein crystals during time-resolved x-ray diffraction experiments. It is a single beam device with a diode array detector for simultaneous multiwavelength measurement of optical spectra. The time resolution of the microspectrophotometer is 2.5 ms, which matches the ms time resolution of the x-ray experiments obtained at existing synchrotron sources. Optical densities of single crystals can be reliably measured up to 2.5 OD, with the linear range extending to 2 OD. The tight focusing of the light beam allows monitoring of crystals as small as 30-50 μm, an important feature since successful reaction initiation and optical monitoring of crystals in time-resolved x-ray diffraction experiments often require very small crystals. The compact design of the microspectrophotometer and the long objective-to-crystal distance make it compatible with the crowded environment of time-resolved x-ray diffraction experiments.