A fully remote capable single crystal UV-Vis microspectrophotometer has been developed at SSRL to monitor the photo-reduction of catalytic metal centers in enzyme active sites, follow enzymatic reactions and characterize protein-ligand complexes that contain intrinsic chromophores. Detailed instructions on how to use the system can be found in the Microspec Tab Manual.
The system is comprised of UV solarization-resistant optical fibers, two reflective Newport Schwardchild objectives (Fig. 1), an Ocean Optics QE65000 Spectrum Analyzer and a Hamamatsu light source with deuterium and halogen lamps.
Figure 1. The microspectrophotometer focusing objectives inserted at the sample position. (Click-on to enlarge)
The UV-Vis microspectrophotometer components are motorized and its operation is fully automated. A 5-axis pico-motor stage is used to accurately align the microspectrophotometer objectives to each other (Fig. 2). Three larger stages are used to align the pair of objectives to the sample position.
Figure 2. 5-axis motor stage for alignment of the of the focusing objective stage (Click-on to enlarge)
By default, a ~50 micron light spot at the sample is used,however the system can be configured to provide a smaller ~10 um spot. A Microspec Tab was created in Blu-Ice for control of the spectrometer. Spectra can be collected as a function of time, x-ray dose or interleaved with data collection.
UV-Visible absorption spectroscopy enhanced x-ray crystallography at synchrotron and x-ray free electron laser sources.
Cohen, A.E.; Doukov, T.; Soltis, M.S. Protein Pept Lett 23, 283-90 (2016).
Crystallographic and single-crystal spectral analysis of the peroxidase ferryl intermediate
Meharenna, Y.T.; Doukov, T.; Li, H.; Soltis, M.; Poulos, T.L. Biochemistry 49, 2984 - 2986 (2010).