Femtosecond Pulse Shaping Enables Rapid Two-Dimensional Infrared Spectroscopy

Jul 01, 2013
Volume 28, Issue 7

Why is pulse shaping better than the other potential solutions — hole-burning and four-wave mixing?

Zanni: One of the neat things about 2D IR via pulse shaping is that you can program the pulse sequence to do whatever you want. Our pulse shapers can collect spectra using either a hole-burning or a four-wave mixing approach. In fact, hole burning done with our pulse shaper is better than with etalons (the traditional way of creating the narrow pulses), because you can use a Gaussian- instead of a Lorentzian-shaped pump pulse (which has better resolution). For any pulse sequence we can also use phase cycling, which was not previously possible, which enables a host of new capabilities including the elimination of mechanical chopping, and thus decreases data collection time by a factor of two. Regarding four-wave mixing, the original way of using four-wave mixing involved having all four pulses have independent laser beams. A setup like that could use our pulse shaper — in fact, we do something similar when collecting 3D IR spectra — but that beam geometry has all the problems associated with high resolution and phasing that I mentioned above. Since inventing our pulse shaping method, we have disassembled all of the four-wave mixing setups in my research group.

What limitations does the pulse-shaping method have?

Zanni: The pump-probe beam geometry that the pulse shaper utilizes has been criticized for being less sensitive than a four-beam mixing geometry, but to my knowledge no one has done a quantitative comparison (I wish that I had done one before disassembling our four-wave mixing setup; rebuilding one would take months).

Polarized laser pulses are a very powerful way of probing molecular structure because they can measure the relative orientations between functional groups or can be used to eliminate diagonal peaks from the 2D IR spectra (which sometimes obscure the weaker cross peaks). The standard pulse shaper can do some, but not all polarizations. My research group has also built a polarization pulse shaper that can create any polarization sequence that one wants, although that style of pulse shaper is not yet available commercially through PhaseTech. Perhaps the biggest drawback is the throughput efficiency of the pulse shaper, which is about 25%. In principle this can be 40 or 50%, and improvements are being made, but it turns out that efficiency is a minor drawback. In the last few years it has become straightforward to generate 25–40 µJ of mid-IR commercial laser sources. To put that quantity in perspective, the first 2D IR experiments in 1998 were done with 1 µJ. In my opinion, the benefits of pulse shaping far outweigh the limitations. I am convinced that the productivity of my research group has surged in the past five years due to mid-IR pulse shaping.

One area where you are applying the technique of 2D IR spectroscopy is to the study of the kinetics of protein aggregation, specifically in the study of amyloid proteins involved in type 2 diabetes. What have you been able to see that you could not have seen with other methods?

Zanni: We published a paper in Nature Chemistry last year that I think exemplifies the types of information that we can obtain with our approach better than other approaches (3). Amyloid fiber formation is an extremely difficult problem for X-ray crystallography and NMR spectroscopy because solving the problem requires information about both structure and kinetics. In this Nature Chemistry paper, we studied a model peptide inhibitor from rodents. The rodent peptide does not aggregate and so it was used to design a drug that was approved by the US Food and Drug Administration. We thought that the rodent peptide would inhibit amylin aggregation by breaking up the C-terminal beta-sheet of the fibers, and would be otherwise inert. Instead, it prevented the N-terminal sheet from forming and ultimately itself templated into beta-sheet fibers onto the side of the human fibers. This rodent peptide had never before been observed to form amyloid fibers. In fact, neither of the usual methods for studying amyloid structures, TEM and Tht fluorescence, revealed any structural changes. Thus, not only were the results surprising, but it exemplified the information content available from 2D IR that is not easily obtained with other methods.

How did your method enable you to see what you saw?

Zanni: We were able to make these novel insights because of three capabilities made possible by 2D IR spectroscopy. First, just as with FT-IR spectroscopy, we can study aggregates, membrane proteins, and other systems that are not easily amenable to X-ray crystallography or NMR spectroscopy. Second, our pulse-shaping technology collects data so quickly that we can monitor kinetics on the fly. That enables us to monitor the real-time aggregation of these proteins. Third, we get good structural information. In our amyloid studies we usually also use isotope labeling, in which case we can obtain residue level structural information on a kinetically evolving system of an aggregate. In this regard there are few, if any, other comparable techniques.


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