Nonlinear Optical Methods for Noninvasive Analytics.

To date, many of the current tools and technologies used to explore biophysical processes and phenomena are limited in part by their inability to probe without external perturbation. Factors such as size restrictions, samples used for non-terminal studies, and small molecule dynamics cannot be addressed by these techniques. This has not only driven the field of noninvasive analytics, but has had a direct impact on shaping the next generation of biological and clinical assays. Highlights of my thesis work herein focus on the development of nonlinear optical spectroscopy and imaging modalities, sum frequency generation (SFG) spectroscopy and coherent anti-Stokes Raman scattering (CARS) microscopy. Each has their own unique qualities that make them ideal as noninvasive tools and techniques. Using these two techniques, my studies address fundamental questions about: (1) the orientation and behavior of chemically immobilized peptides on abiotic surfaces for the rational design of improved biosensors and bioactive textiles, (2) understanding the relations between cytosolic lipids, cellular energy homeostasis/consumption, and developmental biology in female reproductive cells, and (3) the ability to classify and measure male reproductive health as it relates to acrosome integrity. Results from SFG, a surface-sensitive spectroscopy, demonstrate that surface tethering mechanisms govern both the orientation and activity of antimicrobial peptide MSI-78 on surfaces. Attachment of the n-terminus of this peptide results in an orientation perpendicular to the surface normal (lying down) and higher antimicrobial activity whereas c-terminus attachment leads to a parallel orientation (standing up) and lower activity. Other methods complementary to SFG including circular dichroism (CD) spectroscopy and coarse-grained simulation molecular dynamics simulations also support this conclusion. CARS, a live cell noninvasive microscopy, is used to evaluate the contributions of lipid in oocyte growth, development, and for metabolic disease. Results show that lipid content fluctuates as oocytes progress through meiosis, indicated by an increase in content as the oocytes grow and a decrease as oocytes resume meiosis. Lipid content is also higher for oocytes from females who exhibit metabolic disease. As for male reproductive cells, CARS microscopy is beginning to be used for the identification of acrosome reaction, an important predictor of male infertility.PhDBiophysicsUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/113659/1/jasensja_1.pd

Multireflection Sum Frequency Generation Vibrational Spectroscopy

We developed a multireflection data collection method in order to improve the signal-to-noise ratio (SNR) and sensitivity of sum frequency generation (SFG) spectroscopy, which we refer to as multireflection SFG, or MRSFG for short. To achieve MRSFG, a collinear laser beam propagation geometry was adopted and trapezoidal Dove prisms were used as sample substrates. An in-depth discussion on the signal and SNR in MRSFG was performed. We showed experimentally, with “<i>m</i>” total internal reflections in a Dove prism, MRSFG signal is ∼<i>m</i> times that of conventional SFG; SNR of the SFG signal-to-background is improved by a factor of ><i>m</i>^1/2 and <<i>m</i>. MRSFG also improved the SFG sensitivity to resolve weak vibrational signals. Surface molecular structures of adsorbed ethanol molecules, polymer films, and a lipid monolayer were characterized using both MRSFG and conventional SFG. Molecular orientation information on lipid molecules with a 9% composition in a mixed monolayer was measured using MRSFG, which showed a good agreement with that derived from 100% lipid surface coverage using conventional SFG. MRSFG can both improve the spectral quality and detection limit of SFG spectroscopy and is expected to have important applications in surface science for studying structures of molecules with a low surface coverage or less ordered molecular moieties

Molecular Orientation Analysis of Alkyl Methylene Groups from Quantitative Coherent Anti-Stokes Raman Scattering Spectroscopy

Quantitative data analysis in coherent anti-Stokes Raman scattering (CARS) spectroscopy is important for extracting molecular structural information. We developed a method to derive molecular tilt angle with respect to the surface normal based on quantitative CARS spectral analysis. We showed that the tilt angle of methylene alkyl chains on a surface can be directly obtained from the CH₂ symmetric/asymmetric peak ratio in a CARS spectrum. The lipid alkyl chain tilt angle from a lipid monolayer was measured to be ∼0° and was verified by sum frequency generation spectroscopy, which probes the orientations of the lipid methyl end groups. The tilt angle of a silane monolayer alkyl chain was derived to be ∼35°, which agrees with the theoretical prediction. This method is submonolayer sensitive and can also be used to interpret polarization-dependent signals in CARS microscopy. It can be applied to elucidate detailed molecular structure from CARS spectroscopic and microscopic measurements

Quantitative Spectral Analysis of Coherent Anti-Stokes Raman Scattering Signals: C–H Stretching Modes of the Methyl Group

Coherent anti-Stokes Raman scattering (CARS) vibrational spectroscopy has been extensively developed into a powerful analytical technique to study various molecules. Quantitative interpretation of CARS spectra can help to improve CARS for chemical analysis and extend its analytical applications. In this work, we quantitatively analyzed CARS signals originating from the methyl groups in poly­(dimethylsiloxane) (PDMS), with the help of the bond additivity method. Experimentally, a home-built CARS spectrometer modified from a commercial sum frequency generation spectrometer was used to collect CARS spectra from a PDMS film. Theoretically, we successfully reproduced the peak intensity ratio of C–H symmetric and asymmetric stretching modes of the PDMS methyl group in different polarization combinations based on bond additivity method and Raman depolarization ratio. This research shows that bond additivity theory can help to obtain the third-order nonlinear susceptibility tensor properties probed by different polarization combinations used in CARS spectroscopy. The method developed in this work could also be applied to CARS vibrational stretching analysis of other functional groups, providing quantitative understanding of CARS spectrum for applications in spectroscopy

Monitoring Antimicrobial Mechanisms of Surface-Immobilized Peptides in Situ

Antimicrobial peptides (AMPs) in free solution can kill bacteria by disrupting bacterial cell membranes. Their modes of action have been extensively studied, and various models ranging from pore formation to carpet-like mechanisms were proposed. Surface-immobilized AMPs have been used as coatings to kill bacteria and as sensors to capture bacteria, but the interaction mechanisms of surface-immobilized AMPs and bacteria are not fully understood. In this research, an analytical platform, sum frequency generation (SFG) microscope, which is composed of an SFG vibrational spectrometer and a fluorescence microscope, was used to probe molecular interactions between surface-immobilized AMPs and bacteria in situ in real time at the solid/liquid interface. SFG probed the molecular structure of surface-immobilized AMPs while interacting with bacteria, and fluorescence images of dead bacteria were monitored as a function of time during the peptide–bacteria interaction. It was believed that upon bacteria contact, the surface-immobilized peptides changed their orientation and killed bacteria. This research demonstrated that the SFG microscope platform can examine the structure and function (bacterial killing) at the same time in the same sample environment, providing in-depth understanding on the structure–activity relationships of surface-immobilized AMPs

Effect of Solvent on Surface Ordering of Poly(3-hexylthiophene) Thin Films

Enhancement of charge transport in organic polymer semiconductors is a crucial step in developing optimized devices. A variety of sample preparation conditions, such as film fabrication method, solvent species, and annealing, were found to influence the hole mobility of organic polymers. Despite the fact that many factors can influence their performance, it is believed that polymer surface ordering plays a key role in determining organic polymer function. Here, sum frequency generation (SFG) vibrational spectroscopy was used to nondestructively map the surface/interfacial ordering of poly­(3-hexylthiophene) (P3HT) films prepared using different solvents; we believe that solvent interactions determine the degree of surface/interfacial ordering. Both X-ray diffraction (XRD) spectroscopy and scanning electron microscopy (SEM) were used to supplement SFG to systematically study bulk crystallinity and surface morphology. We conclude that SFG is a powerful tool to elucidate the surface/interfacial structural information on polymer semiconducting films. We demonstrate that the solvent composition used to prepare P3HT thin films influences the resulting film surface morphology, surface/interfacial ordering, and bulk crystallinity

Capsaicin-Inspired Thiol–Ene Terpolymer Networks Designed for Antibiofouling Coatings

Novel photocurable ternary polymer networks were prepared by incorporating <i>N</i>-(4-hydroxy-3-meth­oxy­benzyl)-acrylamide (HMBA) into a cross-linked thiol–ene network based on poly­(ethylene glycol)­diacrylate (PEGDA) and (mer­capto­propyl)­methyl­siloxane homopolymers (MSHP). The ternary network materials displayed bactericidal activity against <i>Escherichia coli</i> and <i>Staphylococcus aureus</i> and reduced the attachment of marine organism <i>Phaeodactylum tricornutum</i>. Extensive soaking of the polymer networks in aqueous solution indicated that no active antibacterial component leached out of the materials, and thus the ternary thiol–ene coating killed the bacteria by surface contact. The surface structures of the polymer networks with varied content ratios were studied by sum frequency generation (SFG) vibrational spectroscopy. The results demonstrated that the PDMS Si-CH₃ groups and mimic-capsaicine groups are predominantly present at the polymer–air interface of the coatings. Surface reorganization was apparent after polymers were placed in contact with D₂O: the hydrophobic PDMS Si-CH₃ groups left the surface and returned to the bulk of the polymer networks, and the hydrophilic PEG chains cover the polymer surfaces in D₂O. The capasaicine methoxy groups are able to segregate to the surface in an aqueous environment, depending upon the ratio of HMBA/PEGDA. SFG measurements in situ showed that the antibacterial HMBA chains, rather than the nonfouling PEG, played a dominant role in mediating the antibiofouling performance in this particular polymer system

Observing a Model Ion Channel Gating Action in Model Cell Membranes in Real Time in Situ: Membrane Potential Change Induced Alamethicin Orientation Change

Ion channels play crucial roles in transport and regulatory functions of living cells. Understanding the gating mechanisms of these channels is important to understanding and treating diseases that have been linked to ion channels. One potential model peptide for studying the mechanism of ion channel gating is alamethicin, which adopts a split α/3₁₀-helix structure and responds to changes in electric potential. In this study, sum frequency generation vibrational spectroscopy (SFG-VS), supplemented by attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR), has been applied to characterize interactions between alamethicin (a model for larger channel proteins) and 1-palmitoyl-2-oleoyl-<i>sn</i>-glycero-3-phosphocholine (POPC) lipid bilayers in the presence of an electric potential across the membrane. The membrane potential difference was controlled by changing the pH of the solution in contact with the bilayer and was measured using fluorescence spectroscopy. The orientation angle of alamethicin in POPC lipid bilayers was then determined at different pH values using polarized SFG amide I spectra. Assuming that all molecules adopt the same orientation (a δ distribution), at pH = 6.7 the α-helix at the N-terminus and the 3₁₀-helix at the C-terminus tilt at about 72° (θ₁) and 50° (θ₂) versus the surface normal, respectively. When pH increases to 11.9, θ₁ and θ₂ decrease to 56.5° and 45°, respectively. The δ distribution assumption was verified using a combination of SFG and ATR-FTIR measurements, which showed a quite narrow distribution in the angle of θ₁ for both pH conditions. This indicates that all alamethicin molecules at the surface adopt a nearly identical orientation in POPC lipid bilayers. The localized pH change in proximity to the bilayer modulates the membrane potential and thus induces a decrease in both the tilt and the bend angles of the two helices in alamethicin. This is the first reported application of SFG to the study of model ion channel gating mechanisms in model cell membranes