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Contributions to Science

1. Invention of Alternating Laser Excitation for single molecule FRET method (ALEX, smFRET). Shimon Weiss, Achillefs Kapanidis, and I developed the alternating laser excitation methodology for experiments where single molecule FRET was not able to distinguish between bound, low FRET efficiency species with both donor and acceptor present from species with only donor or acceptor. In order to distinguish between these two situations, we developed the use of alternating laser excitation to determine the presence of the donor and acceptor independently at the single molecule level. The use of ALEX has grown in popularity and use for the last 13 years. We extended the methodology to use fluorescence lifetime information, extracting information about changes in the distance distributions within the unfolded state of the protein Chymotrypsin Inhibitor 2 as the denaturant concentration was decreased. a. A. N. Kapanidis, N. K. Lee, T. A. Laurence, S. Doose, E. Margeat, and S. Weiss, “Fluorescence-aided molecule sorting: analysis of structure and interactions by alternating-laser excitation of single molecules,” Proceedings of the National Academy of Sciences of the United States of America 101, 8936–8941 (2004). b. T. A. Laurence, X. Kong, M. Jäger, and S. Weiss, “Probing structural heterogeneities and fluctuations of nucleic acids and denatured proteins,” Proceedings of the National Academy of Sciences of the United States of America 102, 17348–17353 (2005). c. N. Kapanidis, T. A. Laurence, N. K. Lee, E. Margeat, X. Kong, and S. Weiss, “Alternating-Laser Excitation of Single Molecules,” Accounts of Chemical Research 38, 523–533 (2005).

2. Developed method for evaluating uniformity of plasmonic nanoparticles in solution on a particle-by-particle basis, showing the benefits of rational design of nanoparticles for SERS. The highest SERS signals often occur from randomly aggregated nanoparticles. There have been considerable efforts in designing nanoparticles with reproducible, high SERS enhancements for applications. I developed SERS detection methods for freely diffusing nanoparticles to rapidly detect and characterize distributions of SERS enhancement factors. My Collaborators and I showed that engineered nanoparticles and nanoparticle assemblies were able to improve upon reproducibility while retaining high enhancement. a. T. A. Laurence, G. Braun, C. Talley, A. Schwartzberg, M. Moskovits, N. Reich, and T. Huser, “Rapid, Solution-Based Characterization of Optimized SERS Nanoparticle Substrates,” Journal of the American Chemical Society 131, 162–169 (2009). b. T. A. Laurence, G. B. Braun, N. O. Reich, and M. Moskovits, “Robust SERS Enhancement Factor Statistics Using Rotational Correlation Spectroscopy,” Nano Letters 12, 2912–2917 (2012). c. G. B. Braun, S. J. Lee, T. Laurence, N. Fera, L. Fabris, G. C. Bazan, M. Moskovits, and N. O. Reich, “Generalized Approach to SERS-Active Nanomaterials via Controlled Nanoparticle Linking, Polymer Encapsulation, and Small-Molecule Infusion,” The Journal of Physical Chemistry C 113, 13622–13629 (2009).

3. Enhancement and application of fluorescence correlation spectroscopy methods to protein interactions and aggregation. Fluorescence correlation spectroscopy is a method related to single molecule fluorescence spectroscopy that is well-suited to monitoring strengths of molecular interactions. I have helped determine molecular dynamics, stoichiometry, and binding of several protein interactions since arriving at LLNL. I have developed several methods suited to a variety of specific situations. For example, using what we called “purified FCS”, we found that the DNA sliding clamp of E. coli binds to the 3’ end in the presence of only SSB. In other cases, we measured the binding constant of Y. pestis effectors YopB (a membrane protein) and LcrV (soluble protein) and measured the effects of apoE on amyloid-beta aggregation. We also demonstrated the acquisition of FCS with good signal to noise at very high concentrations (38 µM) of chromophores in standard confocal microscopy. This will have applications in monitoring molecular interactions in cellular environments and for low-affinity, transient molecular interactions. a. T. A. Laurence, Y. Kwon, A. Johnson, C. W. Hollars, M. O’Donnell, J. A. Camarero, and D. Barsky, “Motion of a DNA Sliding Clamp Observed by Single Molecule Fluorescence Spectroscopy,” Journal of Biological Chemistry 283, 22895–22906 (2008). b. S. Ly, F. Bourguet, N. O. Fischer, E. Y. Lau, M. A. Coleman, and T. A. Laurence, “Quantifying Interactions of a Membrane Protein Embedded in a Lipid Nanodisc using Fluorescence Correlation Spectroscopy,” Biophysical Journal 106, L05–L08 (2014). c. S. Ly, R. Altman, J. Petrlova, Y. Lin, S. Hilt, T. Huser, T. A. Laurence, and J. C. Voss, “Binding of Apolipoprotein E Inhibits the Oligomer Growth of Amyloid-beta Peptide in Solution as Determined by Fluorescence Cross-correlation Spectroscopy,” Journal of Biological Chemistry 288, 11628–11635 (2013). d. T. A. Laurence, S. Ly, F. Bourguet, N. O. Fischer, and M. A. Coleman, “Fluorescence Correlation Spectroscopy at Micromolar Concentrations without Optical Nanoconfinement,” The Journal of Physical Chemistry B 118, 9662–9667 (2014).

4. Discovered source and mechanism for laser-induced damage of optical materials on the National Ignition Facility. With 1.8 MJ of energy at 351 nm, the National Ignition Facility is the largest, highest energy laser in the world. It is dedicated to several national security missions, including obtaining ignition of nuclear fusion. During construction, it was clear that, with the then current technology, laser-induced damage of optics would prevent NIF from reaching the design energy of 1.8 MJ. Defects on or near the surfaces of fused silica optics caused laser damage, but their identity and depth in the material were unclear. Since absorbing defects will re-emit fluorescence at some level, we searched for defects using high-sensitivity fluorescence lifetime imaging (as used in single molecule studies). We found defects were only at the surface, and that they could be created in the fused silica material itself without contaminants via fracture. These small fractures were caused in the final polishing steps in optical finishing, and we etched open the fractures using a buffered oxide etch (BOE). After extensive development, the etching process significantly lower laser-induced damage in NIF optics, allowing it to reach the design energy. Of interest to this proposal, the fluorescence lifetime imaging effort supported some of the algorithm development we will include in the open source software, and this effort supports the significant maintenance and acquisition costs of the microscopy equipment in my lab. a. T. A. Laurence, J. D. Bude, N. Shen, T. Feldman, P. E. Miller, W. A. Steele, and T. Suratwala, “Metallic-like photoluminescence and absorption in fused silica surface flaws,” Applied Physics Letters 94, 151114 (2009). b. T. I. Suratwala, P. E. Miller, J. D. Bude, W. A. Steele, N. Shen, M. V. Monticelli, M. D. Feit, T. A. Laurence, M. A. Norton, C. W. Carr, and L. L. Wong, “HF-Based Etching Processes for Improving Laser Damage Resistance of Fused Silica Optical Surfaces,” Journal of the American Ceramic Society 94, 416–428 (2011). c. T. A. Laurence, J. D. Bude, S. Ly, N. Shen, and M. D. Feit, “Extracting the distribution of laser damage precursors on fused silica surfaces for 351 nm, 3 ns laser pulses at high fluences (20-150 J/cm2),” Optics Express 20, 11561–11573 (2012). d. S. Ly, N. Shen, R. A. Negres, C. W. Carr, D. A. Alessi, J. D. Bude, A. Rigatti, and T. A. Laurence, “The role of defects in laser-induced modifications of silica coatings and fused silica using picosecond pulses at 1053 nm: I Damage morphology,” Optics Express 25, 15161 (2017).