Instrument and Software Development

Stopped-flow Infrared Spectroscopy 

At the John Innes Centre, and at Lawrence Berkeley National Laboratory, I built a stopped-flow infrared instrument specifically for biological chemistry in aqueous solutions.

Our achievement is to measure good time-resolved IR spectra in a stopped-flow experiment with catalytically appropriate concentrations of protein (~ 50 µM).  The infrared (IR) spectrum of a protein provides information about the polypeptide backbone and organic and metal cofactors.  In particular, small molecules (NO, N2, CN-, CO, NO2-, N3-, O2-) bound to biological metal sites are of particular interest as these often yield sharp well defined infrared bands.  Time resolution adds the ability to measure kinetic and chemical data on the binding and catalytic transformation of substrates, and concomitant conformational changes in the protein matrix.  

The SF-IR instrument comprises a Bruker IFS 66/S FTIR interfaced to an anaerobic/dry glove box (Belle Technology) with a home-built flow system and a home-built 50 µm pathlength cuvette with an integrated mixer.  Inline optical filters are used to enhance signal-to-noise.  This setup gives us excellent performance.  At 2000 cm-1, for a 50 µM aqueous sample (a background of 0.6 A), we achieve a peak-to-peak noise level of better than 0.0001 A for a 27 ms scan at 4 cm-1 resolution.  The baseline stability between stopped-flow samples generally within 0.00006 A.  This noise and stability performance means that experiments where a single CO is binding to a metalloprotein centre over a second require about 50 µM samples (after mixing).  This signal to noise level can, of course, be enhanced by co-adding several stopped-flow experiments. Also included is a multi-mixing attachment and the simultaneous measurement of UV-visible kinetics.  

I have plans for the next generation of SF-IR instrumentation, including a new cuvette design for smaller volume samples, better multi-mixing, possible incorporation of microfluidics and the ability to perform flow-flash experiments.  

Soft X-ray Spectroscopy (Soft XAS)

For much of the past 30 years, I have both lead and contributed to the design, construction and daily operation of hardware for synchrotron beamlines, including high-vacuum chambers, liquid helium cryostats, superconducting magnets, and specialized sample preparation chambers.  Much of this equipment measured soft X-ray (50 - 2000 eV) spectra of dilute materials.  Soft X-ray spectra can have a high information content, however, soft X-ray measurements are much more technically challenging than higher energy “hard” X-ray experiments.

I performed this work while working at the National Synchrotron Light Source (NSLS) in New York, Stanford Synchrotron Radiation Lightsource (SSRL), U.C Davis and Lawrence Berkeley National Laboratory (LBNL).  At LBNL, I was a senior scientist and day-to-day manager of ABEX (Advanced Biological and Environmental X-ray) facility; a DOE OBER funded User facility which used soft X-rays to characterize complex biological and environmental systems. 

The figure shows a montage of some of the X-ray beamline equipment I have worked with. Of particular interest is the chamber for low-temperature X-ray Magnetic Circular dichroism spectroscopy which was capable of 6 Tesla magnetic field, sample temperatures down to 2 K, and allowed ready anaerobic exchange of samples, including frozen materials.