New Developments in 2D IR Advance Medical Research and Materials Analysis

Coherent two-dimensional infrared spectroscopy (2D IR) uses a series of IR femtosecond laser pulses to pump and then probe the response of a system, making it possible to learn much more about the structure and dynamics of molecules than can be seen with one-dimensional IR spectroscopy. The technique’s inventor, Martin T. Zanni of the University of Wisconsin-Madison, discussed 2D IR in a 2013 interview in Spectroscopy (1).  Since 2013, Zanni has applied 2D IR spectroscopy to new systems and has started a company, PhaseTech Spectroscopy, Inc., to commercialize the technique. Zanni will receive the 2017 Craver Award from the Coblentz Society for this work. This interview is part of a series of interviews with the winners of awards that will be presented at the SciX 2017 conference in October.

Can you please give us an update on your research since 2013 (1)?

Our 2013 interview in Spectroscopy provided an overview of coherent two-dimensional infrared (2D IR) spectroscopy and how the mid-IR pulse shaping technology we had developed made it more robust, and discussed some of our work using 2D IR on type 2 diabetes and some simple solar cells. We have covered a lot of new ground since then. On the diabetes project, we discovered a cluster of proteins that we think may be responsible for killing the beta cells in the pancreases of people who have type 2 diabetes, making them more dependent on insulin injections. We started a new line of research studying cataracts, which is some of the first tissue work using 2D IR spectroscopy. In addition, we built the first wide-field 2D IR microscope so that we can now start imaging using the hyper spectral information content of 2D IR spectra. The materials side of my group has now really expanded. For materials, we have transferred some of our technologies from the mid-IR to the visible, and generated a new variant of 2D visible spectroscopy that we call 2D white-light spectroscopy. Perhaps our most impactful accomplishment since 2013 was to start PhaseTech. We have now sold 2D IR and 2D visible spectrometers across the globe. 

How did 2D IR help you make your diabetes discovery?

Almost all people who contract type 2 diabetes have amyloid fibers in their pancreas, where insulin is created. Mysteriously, if amylin fibers are mixed with pancreas beta-cells in a cell culture, the cells are perfectly happy and go on making insulin. If instead, the amylin protein is mixed with cells after the protein has had a chance to age, but not aged quite enough to make fibers, then it is extremely toxic to beta-cells. As a result, lots and lots of people are trying to figure out how the proteins preassemble into something so toxic. We used 2D IR spectroscopy to watch the self-assembly of amylin proteins into amyloid fibers. In a test tube, we used our mid-IR pulse-shaping version of 2D IR spectroscopy to watch the fibers form and discovered that before they aggregate into fibers, they first self-assemble into a cluster of proteins in which the center segment of their sequence lines up into parallel beta-sheets (2). It was fascinating to see this happen because it is very well known that the center sequence, which is formed by the amino acids FGAIL, is critical to diabetes. Humans get diabetes and so do other mammals, such as cats, but lots of other mammals do not, such as mice, rats, and bears. The FGAIL sequence is slightly different from mammal to mammal. Our results suggest that in some species, differences in this sequence prevent this cluster of proteins from forming, thereby preventing the toxic species. There are many more experiments that we must do to further test this hypothesis, but it is an intriguing and potentially important realization.

Was it difficult to apply to coherent 2D IR spectroscopy to tissues in the cataracts work?

It was surprisingly easy! We had anticipated that the tissues would scatter the laser light horribly, producing a large background, but instead the spectra turned out to be very straightforward to measure. It has been fascinating. In our first published paper (3), we irradiated lenses dissected from the eyeballs of pigs with ultraviolet (UV) light and measured the 2D IR spectra. It turns out that we saw the beta-sheet structures typical of amyloid fibers! We looked very hard with different imaging techniques, but could not see fibers. About 40% of the proteins in the lens are chaperone proteins, suggesting that the amyloid beta-sheet structures are sequestered before they become large enough to see in images. It is fascinating. Work is underway now on human lens tissues. 

What was involved in applying the concept of 2D spectroscopy to the visible region of the spectrum?

The biggest hurdle for us in switching to the visible was conceptual. There are lots of groups utilizing 2D visible spectroscopies: 2D vis spectroscopy is essentially the same technique as 2D IR spectroscopy, albeit at a different wavelength. However, besides wavelength, there is another big difference, which is that visible linewidths are much, much, broader than IR linewidths. Visible linewidths can span hundreds of nanometers. It is very difficult to generate intense laser pulses of comparable width. If the bandwidth of your laser pulse is not at least as broad, if not broader, than the linewidth itself, then you lose all the advantages of 2D spectroscopy because you can’t see the cross peaks or 2D lineshapes. We did not enter the field until we had a new idea, which was to use continuum generation for the pulse sequences. Continuum generation has been used as a probe pulse for decades, but it is very weak, which is my guess why no one had tried to use it as a pump pulse before. We generated an entire 2D pulse sequence with it, and it worked great. White light from continuum generation spans nearly the entire visible and near-infrared, and so we could then collect 2D spectra, which we now call 2D white-light spectra, with bandwidth that exceeds even the broadest transitions (4). We are now measuring a variety of semiconducting materials using this new source.

What has it been like commercializing coherent 2D spectroscopies?

It has been very satisfying. My company has helped expand the field quite significantly over the four years that it has been in operation. It has significantly lowered the hurdle for nonexperts to join the 2D field, and made it much faster for experienced users to incorporate pulse shaping into their 2D experiments. It has also given me more of an engineering perspective when we design a new instrument in my research group, because simple instruments are more robust, which leads to higher productivity. It is an exciting time for 2D spectroscopy, because new laser sources are becoming available that will expand the applicability of the technique and lower the cost. 

References

• L. Bush, Spectroscopy28(7), 24–30 (2013).http://www.spectroscopyonline.com/femtosecond-pulse-shaping-enables-rapid-two-dimensional-infrared-spectroscopy?id=&pageID=1&sk=&date=

• L.E. Buchanan, E.B. Dunkelberger, H.Q. Tran, P.-N. Cheng, C.-C. Chiu, P. Cao, D.P. Raleigh, J.J. de Pablo, J.S. Nowick, and M.T. Zanni, PNAS110, 19285 (2013).

• T.O. Zhang, A.M. Alperstein, and M.T. Zanni, J. Mol. Biol. in press, accepted manuscript (2017).

• R.D. Mehlenbacher, T.J. McDonough, M. Grechko, M.-Y. Wu, M.S. Arnold, and M.T. Zanni, Nature Comm. 6, 6732 (2015).