SPECTROSCOPY: Biospectroscopy uses mid-IR pulse shaper

Two-dimensional (2-D) IR spectroscopy is a powerful tool for studying biomolecular structures and their dynamics. However, traditional methods of collecting 2-D IR spectra are often difficult to implement, cause distorted peak shapes, and have poor temporal resolution and/or phase problems. By using a programmable mid-IR pulse shaper based on a germanium acousto-optic modulator, researchers in the Department of Chemistry at the University of Wisconsin (Madison, WI) have overcome many of these obstacles and have been able to apply this improved 2-D IR spectroscopy technique to the study of fiber structures in aggregated human islet amyloid polypeptide (hIAPP), which is involved with type-2 diabetes in humans.1


A 2-D mid-IR pulse shaper can be used to obtain improved infrared spectra of biological materials. A Fourier-transform infrared (FTIR) trace (top) is compared to a 2-D spectral plot of the fibrous structure of a polypeptide that is commonly involved with type-2 diabetes. (Courtesy of University of Wisconsin)
Click here to enlarge image

A typical 2-D IR spectrometer is a complicated instrument because individual pulses have to be routed down different optical paths using mirrors and mechanical stages to control their time delays by changing the physical length of the optical path. Even though all the pulses have the same shape and frequency (unless additional optics such as etalons are added to narrow the pulse bandwidth, or a second mixing crystal is used to generate pulse sequences with two center frequencies), implementation of a simple pulse sequence is difficult because four mid-IR pulses are required, all of which are invisible to the naked eye. Aligning four invisible laser beams is technically challenging and in stark contrast to the well-established technique of 2-D nuclear-magnetic-resonance (NMR) spectroscopy, in which computerized electronics make it straightforward to implement pulse sequences with specified frequencies, time delays, phases, and intensities simply by programming. Automation of 2-D IR pulse sequences is necessary to utilize the full capabilities of this unique, ultrafast time-resolution 2‑D IR spectroscopy technique.

Controlled phases, delays, and shapes

To automate 2-D IR spectroscopy and make its implementation more like 2-D NMR spectroscopy, the researchers used a mid-IR pulse shaper to electronically generate 2-D IR pulse sequences with controlled phases, delays, and shapes. These sequences mimic the two common methods for obtaining 2-D IR spectra: “hole burning,” or using an etalon to scan a narrowband mid-IR pulse across a protein’s absorption spectrum and plotting vibrational-mode intensity changes; and pulsed 2-D IR spectroscopy that uses femtosecond mid-IR pulses to excite a sample, the photon echo of which can be measured and Fourier-transformed to obtain the spectra.

For the hole-burning method, the germanium acousto-optic mid-IR pulse shaper takes a femtosecond pulse and tailors its phase and amplitude to produce a frequency-narrowed pump pulse. And for the pulsed method, one of two beams passes through the shaper creating a femtosecond pulse pair, a collinear method in which the time delays are perfectly set, allowing the automatic generation of absorptive 2-D IR spectra with proper phases and ultrafast time resolution.

To demonstrate the advantages of automated 2-D IR spectroscopy, the researchers measured the spectrum of hIAPP peptides that are involved in the disease mechanism of type-2 diabetes. These peptides aggregate to form long fibers and are difficult to study with traditional 2-D IR techniques because they scatter light. But by phase-cycling the pump pulses (a method now made possible by the programmable shaper), the observed frequency of the scatter is shifted away from the desired signal, leaving a high-resolution spectrum of the hIAPP fibers (see figure). The spectrum reflects the structural disorder of the fibers, which are an important characteristic of the disease mechanism.

“The key to automating these new and exciting 2-D IR spectroscopies was to develop a mid-IR pulse shaper, which is the first of its kind,” said principal investigator Martin Zanni. “While we demonstrated our approach on a very interesting problem associated with diabetes, perhaps the greatest achievement at this time is the automation of the device itself. In time, automated 2-D IR spectroscopy will become a common analytical technique, widely available in university and industrial research laboratories around the world.”

Gail Overton

REFERENCE

1. S-H Shim et al., Proc. National Academy of Sciences 10.1073, pnas.0700804104, 1 (May 2007).

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