Understanding the way that proteins and other biological molecules operate means digging deeply -- examining not only their structures, but also seeing how they change shape under various conditions.Nuclear magnetic resonance and x-ray crystallography can help visualize molecules down nearly to the atomic level, but they provide only static images. And although two-dimensional infrared spectroscopy helps map molecular position and orientation, it does so only for molecules that are in equilibrium.Now scientists at Universität Zürich in Switzerland and at Ruhr-Universität Bochum in Germany have devised a technique that expands on two-dimensional IR spectroscopy to provide real-time imaging of molecular conformational changes. They report their work in the Nov. 23 issue of Nature.The investigators performed their technique, dubbed transient 2-D IR spectroscopy, using as their subject a loop of several amino acids held stably in a bow-shaped conformation by an internal disulfide bond.They used a 790-nm Ti:sapphire laser made by Spectra-Physics that was frequency-tripled to 266 nm to cleave the disulfide bond, causing the molecule to unfold and relax. After a delay of 3, 25 or 100 ps, they recorded the 2-D IR spectrum as the peptide changed shape, acquiring two sets of data simultaneously. They used a monochromator made by Jobin Yvon and a 32-pixel HgCdTe detector made by Infrared Associates Inc. of Stuart, Fla., to collect the spectral images.The scientists then turned to a “double differencing” analytical technique, subtracting the 2-D IR spectra acquired with the 266-nm pulse on from those taken with the pulse off as well as subtracting spectra acquired with the pump pulses of IR radiation off from those acquired with the pulses on. This enabled them to look beyond the typical properties viewed by 1-D IR spectroscopy -- such as dipole strength -- and to sense the more minute ones that are indicative of bond interactions -- such as couplings between vibrational oscillations. Ultimately, each set of difference spectra provided one frame of a spectroscopic movie of the molecule in motion.They found that molecular motion was visualizable with picosecond resolution, and they saw a hydrogen bond within the peptide stretch without breaking, causing the molecule to shift such that a carbon-oxygen coupling moved to a new position across the hydrogen bond.