Integrated AFM-Raman for molecular characterization of peptide nano- and micro-tubes

This work is focused on exploring a unique integration of techniques, Raman micro-spectroscopy and atomic force microscopy (AFM), which when combined offer more than the sum of their respective parts. The non-invasive chemical specificity afforded by Raman spectroscopy, combined with the nanoscale-resolution topographic imaging of AFM offer much individually. The physics underlying the practical application of each technique is very general; Raman spectroscopy detects molecular vibrational shifts using light, and AFM uses a physical probe to interact with a surface to provide topographic (and mechanical) information. As a result, there are few restrictions to the possible samples that can be studied with these techniques, from semiconductors and geological crystals, through to simple organic chemical structures all the way to complex biological molecules and systems such as cells and tissue. In this work, a synthetic biomaterial composed of diphenylalanine (FF) peptide units which self-assemble into strong tubular structures is used as a sample of interest when exploring the different possibilities available from a combined Raman-AFM instrument. First, the combined system was set up in order to perform tip-enhanced Raman spectroscopy (TERS), a technique promising Raman spectroscopic imaging at the resolution of AFM imaging. A relatively young technique, TERS has huge potential in extending the reach of Raman spectroscopic imaging to the nanoscale, at a regime where a great deal of structure exists, but is usually blurred by conventional diffraction-limited Raman microspectroscopy. A major focus in this work is addressing a current problem with TERS: the fabrication of suitable probes. TERS typically utilizes AFM tips modified to have a silver nanoparticle, capable of locally enhancing the Raman signal, attached at the probe apex. A new method is presented here that promises several improvements over existing approaches, as the entire fabrication can be performed in-situ on the instrument. Tips produced in this way are then characterized by electron microscopy and tested on FF nanotubes. Following this, several techniques for the synthesis of silver nanoparticles are explored for use in TERS. Here, the focus is particularly on decahedral nanoparticles, which can be grown into rod shaped particles with well- defined shapes and sizes. These are important considerations for obtaining the desired enhancing properties for TERS probes. Finally, the AFM-Raman instrument is used to investigate the mechanical properties of FF tubes using several methods. AFM force spectroscopy of tubes suspended across a gap can be used in conjunction with a bending beam theory to measure the Young's modulus of individual tubes. A new type of co-localized experiment using polarized Raman spectroscopy on a suspended tube under various forces from the AFM is tested, and subsequently information relating to the hydrogen bonding network is used, in conjunction with existing X-ray data, to determine the molecular contributions to the modulus using a simple model for amyloid fibrils. Each experiment operates at the single fibril level, with the same fibrils being used, such that different methods can be compared for a single FF tube

DMD-based software-configurable spatially-offset Raman spectroscopy for spectral depth-profiling of optically turbid samples

Spectral depth-profiling of optically turbid samples is of high interest to a broad range of applications. We present a method for measuring spatially-offset Raman spectroscopy (SORS) over a range of length scales by incorporating a digital micro-mirror device (DMD) into a sample-conjugate plane in the detection optical path. The DMD can be arbitrarily programmed to collect/reject light at spatial positions in the 2D sample-conjugate plane, allowing spatially offset Raman measurements. We demonstrate several detection geometries, including annular and simultaneous multi-offset modalities, for both macro- and micro-SORS measurements, all on the same instrument. Compared to other SORS modalities, DMD-based SORS provides more flexibility with only minimal additional experimental complexity for subsurface Raman collection

Integrated AFM-Raman for molecular characterization of peptide nano- and micro-tubes

This work is focused on exploring a unique integration of techniques, Raman micro-spectroscopy and atomic force microscopy (AFM), which when combined offer more than the sum of their respective parts. The non-invasive chemical specificity afforded by Raman spectroscopy, combined with the nanoscale-resolution topographic imaging of AFM offer much individually. The physics underlying the practical application of each technique is very general; Raman spectroscopy detects molecular vibrational shifts using light, and AFM uses a physical probe to interact with a surface to provide topographic (and mechanical) information. As a result, there are few restrictions to the possible samples that can be studied with these techniques, from semiconductors and geological crystals, through to simple organic chemical structures all the way to complex biological molecules and systems such as cells and tissue. In this work, a synthetic biomaterial composed of diphenylalanine (FF) peptide units which self-assemble into strong tubular structures is used as a sample of interest when exploring the different possibilities available from a combined Raman-AFM instrument. First, the combined system was set up in order to perform tip-enhanced Raman spectroscopy (TERS), a technique promising Raman spectroscopic imaging at the resolution of AFM imaging. A relatively young technique, TERS has huge potential in extending the reach of Raman spectroscopic imaging to the nanoscale, at a regime where a great deal of structure exists, but is usually blurred by conventional diffraction-limited Raman microspectroscopy. A major focus in this work is addressing a current problem with TERS: the fabrication of suitable probes. TERS typically utilizes AFM tips modified to have a silver nanoparticle, capable of locally enhancing the Raman signal, attached at the probe apex. A new method is presented here that promises several improvements over existing approaches, as the entire fabrication can be performed in-situ on the instrument. Tips produced in this way are then characterized by electron microscopy and tested on FF nanotubes. Following this, several techniques for the synthesis of silver nanoparticles are explored for use in TERS. Here, the focus is particularly on decahedral nanoparticles, which can be grown into rod shaped particles with well- defined shapes and sizes. These are important considerations for obtaining the desired enhancing properties for TERS probes. Finally, the AFM-Raman instrument is used to investigate the mechanical properties of FF tubes using several methods. AFM force spectroscopy of tubes suspended across a gap can be used in conjunction with a bending beam theory to measure the Young's modulus of individual tubes. A new type of co-localized experiment using polarized Raman spectroscopy on a suspended tube under various forces from the AFM is tested, and subsequently information relating to the hydrogen bonding network is used, in conjunction with existing X-ray data, to determine the molecular contributions to the modulus using a simple model for amyloid fibrils. Each experiment operates at the single fibril level, with the same fibrils being used, such that different methods can be compared for a single FF tube

Holographic optical trapping Raman micro-spectroscopy for non-invasive measurement and manipulation of live cells

We present a new approach for combining holographic optical tweezers with confocal Raman spectroscopy. Multiple laser foci, generated using a liquid-crystal spatial light modulator, are individually used for both optical trapping and excitation of spontaneous Raman spectroscopy from trapped objects. Raman scattering from each laser focus is spatially filtered using reflective apertures on a digital micro-mirror device, which can be reconfigured with flexible patterns at video rate. We discuss operation of the instrument, and performance and viability considerations for biological measurements. We then demonstrate the capability of the instrument for fast, flexible, and interactive manipulation with molecular measurement of interacting live cell systems

Active efflux leads to heterogeneous dissipation of proton motive force by protonophores in bacteria

ABSTRACT Various toxic compounds disrupt bacterial physiology. While bacteria harbor defense mechanisms to mitigate the toxicity, these mechanisms are often coupled to the physiological state of the cells and become ineffective when the physiology is severely disrupted. Here, we characterized such feedback by exposing Escherichia coli to protonophores. Protonophores dissipate the proton motive force (PMF), a fundamental force that drives physiological functions. We found that E. coli cells responded to protonophores heterogeneously, resulting in bimodal distributions of cell growth, substrate transport, and motility. Furthermore, we showed that this heterogeneous response required active efflux systems. The analysis of underlying interactions indicated the heterogeneous response results from efflux-mediated positive feedback between PMF and protonophores’ action. Our studies have broad implications for bacterial adaptation to stress, including antibiotics. IMPORTANCE An electrochemical proton gradient across the cytoplasmic membrane, alternatively known as proton motive force, energizes vital cellular processes in bacteria, including ATP synthesis, nutrient uptake, and cell division. Therefore, a wide range of organisms produce the agents that collapse the proton motive force, protonophores, to gain a competitive advantage. Studies have shown that protonophores have significant effects on microbial competition, host-pathogen interaction, and antibiotic action and resistance. Furthermore, protonophores are extensively used in various laboratory studies to perturb bacterial physiology. Here, we have characterized cell growth, substrate transport, and motility of Escherichia coli cells exposed to protonophores. Our findings demonstrate heterogeneous effects of protonophores on cell physiology and the underlying mechanism

Co-localised Raman and force spectroscopy reveal the roles of hydrogen bonds and ?-? interactions in defining the mechanical properties of diphenylalanine nano- and micro-tubes

An integrated atomic force and polarized Raman microscope were used to measure the elastic properties of individual diphenylalanine (FF) nano- and micro-tubes and to obtain quantitative information regarding the inter-molecular interactions that define their mechanical properties. For individual tubes, co-localised force spectroscopy and Raman spectroscopy measurements allowed the calculation of the Young’s and shear moduli (2565 GPa and 0.2860.05 GPa, respectively) and the contribution of hydrogen bonding network to the Young’s modulus (!17.6 GPa). The p-p interactions between the phenyl rings, dominated by T-type arrangements, were estimated based on previously published X-ray data to only 0.20 GPa. These results provide experimental evidence obtained from individual FF tubes that the network of H-bonds dominates the elastic properties of the FF tubes

Label-free Raman hyperspectral imaging of single cells cultured on polymer substrates

While Raman hyper-spectral imaging has been widely used for label-free mapping of biomolecules in cells, these measurements require the cells to be cultured on weakly Raman scattering substrates. However, many applications in biological sciences and engineering require the cells to be cultured on polymer substrates that often generate large Raman scattering signals. Here, we discuss the theoretical limits of the signal-to-noise ratio in the Raman spectra of cells in the presence of polymer signals and how optical aberrations may affect these measurements. We show that Raman spectra of cells cultured on polymer substrates can be obtained using automatic subtraction of the polymer signals and demonstrate the capabilities of these methods in two important applications: tissue engineering and in-vitro toxicology screening of drugs. Apart from their scientific and technological importance, these applications are examples of the two most common measurement configurations: 1) cells cultured on an optically thick polymer substrate measured using an immersion/dipping objective; 2) cells cultured on a transparent polymer substrate and measured using an inverted optical microscope. In these examples we show that Raman hyperspectral datasets with sufficient quality can be successfully acquired to map the distribution of common biomolecules in cells, such as nucleic acids, proteins and lipids, as well as detecting the early stages of apoptosis. We also discuss strategies for further improvements that could expand the application of Raman hyperspectral imaging on polymer substrates even further in biomedical sciences and engineering

Applications of spatial light modulators in Raman spectroscopy

Advances in consumer display screen technologies have historically been adapted by researchers across the fields of optics as they can be used as electronically controlled spatial light modulators (SLMs) for a variety of uses. The performance characteristics of such SLM devices based on liquid crystal (LC) and digital-micro-mirror device (DMD) technologies in particular has developed to the point where they are compatible with increasingly sensitive instrumental applications, for example Raman spectroscopy. SLMs provide additional flexibility, from modulation of the laser excitation (including multiple laser foci patterns), manipulation of microscopic samples (optical trapping) or selection of sampling volume (adaptive optics or spatially offset Raman spectroscopy), to modulation in the spectral domain for high-resolution spectral filtering or multiplexed/compressive fast detection. Here, we introduce the benefits of different SLM devices as a part of Raman instrumentation, and provide a variety of recent example applications which have benefited from their incorporation into a Raman system