Fluorescence-Encoded Infrared Spectroscopy for Single-Molecule Chemical Dynamics

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In this project supported by the Chemical Structure, Dynamics and Mechanisms-A Program of the Division of Chemistry, Professor Andrei Tokmakoff and his research group at the University of Chicago are developing a new type of optical spectroscopy that can detect single molecules, and provide information about their structure (in other words, the geometric arrangement of atoms in the molecule). Achieving both single molecule detection and structural information in a single experiment is a long-sought-after, but very challenging goal. Professor Tokmakoff's strategy involves the use of both the fluorescence and vibrational properties of molecules. In fluorescence, a molecule exposed to visible or ultraviolet light re-emits light of a specific wavelength range (color). This emission can be very strong, even enough for a single molecule to be detected. However, the emitted light does not tell us very much about the structure of the molecule. On the other hand, molecules vibrate with frequencies that correspond to infrared light ("IR"), what we feel as heat, and also exploit in night vision goggles. Observing how molecules absorb IR light can tell us a lot about their structure, but one needs a high concentration of molecules to obtain sufficient signal. Tokmakoff uses laser pulses of visible and infrared light, carefully sequenced in such a way the intense fluorescence emission contains the information normally provided in an IR experiment, hence the name Fluorescence-Encoded Infrared (FEIR) spectroscopy. The research is focusing the FEIR technique on simple reactions like acid-dissociation, where a molecule loses a proton (positively charged hydrogen atom), and bimolecular complex formation, where two molecules stick to each other and undergo subtle structural changes. FEIR represents an entirely new approach to understanding chemical reactions, and has far -reaching implications not only in chemistry, but materials science and biophysics. The graduate students engaged in this project are experiencing a cross-disciplinary setting that includes design and construction of advanced instrumentation, training in optics and spectroscopy, methods in molecular biology, and molecular modeling and computer simulation. For broad distribution of course materials, Professor Tokmakoff has also made online instructional resources accessible by others in the field. The method used in this project, fluorescence-encoded infrared (FEIR) spectroscopy, combines structural specificity and SM sensitivity by encoding the absorption of IR light onto a visible fluorescence signal. The Tokmakoff group has demonstrated the principles and capabilities of FEIR, using ensemble measurements of ultrafast Fourier-transform and multidimensional spectroscopy, and developed the experimental capability for measuring FEIR spectra and femtosecond transients at 10-100 nanomolar concentrations, approaching the sensitivity regime of individual molecules. The objective of this project is to develop the capabilities for performing an FEIR analog of fluorescence correlation spectroscopy (FCS) experiments, in which the vibrational spectrum is used to track the time-evolving state of molecules for studies of chemical kinetics and dynamics. The Tokmakoff group will apply this technique to the kinetics of proton transfer and bimolecular association/dissociation equilibria, and study how FEIR spectra sense the environment of the fluorescent probe. This is imagined as a first step in the longer-term goal of realizing a method to track the time-evolving structures of single molecules in solution by integrating vibrational information into the real-time photon streams analyzed in SM fluorescence spectroscopy. In addition to the aforementioned training of graduate students, this project supports the continued development and distribution of educational material in the form of continually updated online textbooks and related resources, in the areas of quantum mechanics, spectroscopy, and molecular biophysics.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.