Understanding the way that molecules operate means digging deeply — examining not only their structures, but also how they change shape under various conditions.Techniques such as nuclear magnetic resonance and x-ray crystallography help researchers visualize molecules down nearly to the atomic level, but they provide only a static image. On the other hand, two-dimensional infrared spectroscopy helps map molecular position and orientation, but only for molecules that are in equilibrium.Now scientists at Universität Zürich in Switzerland and at Ruhr-Universität Bochum in Germany together have devised a technique that expands on 2-D IR spectroscopy to provide real-time imaging of molecular conformational changes.Using a cyclic disulfide-bridged peptide — a loop of several amino acids held stably in a bow-shaped conformation by the internal bond — as their subject, the investigators performed their technique, dubbed transient 2-D IR spectroscopy.To initiate conformational changes in the peptide, they used a 790-nm Ti:sapphire laser made by Spectra-Physics that was frequency-tripled to 266 nm to cleave the disulfide bridge, 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.They subtracted the 2-D IR spectra acquired with the 266-nm pulse “on” from those acquired with the UV pulse “off,” revealing difference spectra that are sensitive to changes only during the unfolding process. In turn, spectra acquired during the 2-D IR experiments with the pump pulses of IR radiation off were subtracted from those with the pump pulses on.This “double differencing” technique 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 could visualize molecular motion 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.Nature, Nov. 23, 2006, pp. 469-472.