Probing with randomly interleaved pulse train bridges the gap between ultrafast pump-probe and nanosecond flash photolysis.

Despite the long-standing importance of transient absorption (TA) spectroscopy, many researchers remain frustrated by the difficulty of measuring the nanosecond range in a wide spectral range. To address this shortcoming, we propose a TA spectrophotometer in which there is no synchronization between a pump pulse and a train of multiple probe pulses from a picosecond supercontinuum light source, termed the randomly-interleaved-pulse-train (RIPT) method. For each pump pulse, many monochromatized probe pulses impinge upon the sample, and the associated pump-probe time delays are determined passively shot by shot with subnanosecond accuracy. By repeatedly pumping with automatically varying time delays, a TA temporal profile that covers a wide dynamic range from subnanosecond to milliseconds is simultaneously obtained. By scanning wavelength, this single, simple apparatus acquires not only wide time range TA profiles, but also broadband TA spectra from the visible through the near-infrared regions. Furthermore, we present a typical result to demonstrate how the RIPT method may be used to correct for fluorescence, which often pollutes TA curves.

Development of a rapid Buffer-exchange system for time-resolved ATR-FTIR spectroscopy with the step-scan mode.

Attenuated total reflectance (ATR)-FTIR spectroscopy has been widely used to probe protein structural changes under various stimuli, such as light absorption, voltage change, and ligand binding, in aqueous conditions. Time-resolved measurements require a trigger, which can be controlled electronically; therefore, light and voltage changes are suitable. Here we developed a novel, rapid buffer-exchange system for time-resolved ATR-FTIR spectroscopy to monitor the ligand- or ion-binding re-action of a protein. By using the step-scan mode (time resolution; 2.5 ms), we confirmed the completion of the buffer-exchange reaction within ∼25 ms; the process was monitored by the infrared absorption change of a nitrate band at 1,350 cm(-1). We also demonstrated the anion-binding reaction of a membrane protein, Natronomonas pharaonis halorhodopsin (pHR), which binds a chloride ion in the initial anion-binding site near the retinal chromophore. The formation of chloride- or nitrate-bound pHR was confirmed by an increase of the retinal absorption band at 1,528 cm(-1). It also should be noted that low sample consumption (∼1 µg of protein) makes this new method a powerful technique to understand ligand-protein and ion-protein interactions, particularly for membrane proteins.