Chad E. Talley

Surface-enhanced Raman scattering on nanoporous Au

Colloidal solutions of metal nanoparticles are currently among the most studied substrates for sensors based on surface-enhanced Raman scattering (SERS). However, such substrates often suffer from not being cost-effective, reusable, or stable. Here, we develop nanoporous Au as a highly active, tunable, stable, biocompatible, and reusable SERS substrate. Nanoporous Au is prepared by a facile process of free corrosion of AgAu alloys followed by annealing. Results show that nanofoams with average pore widths of ∼250nm exhibit the largest SERS signal for 632.8nm excitation. This is attributed to the electromagnetic SERS enhancement mechanism with additional field localization within pores.

High resolution fluorescence imaging with cantilevered near‐field fiber optic probes

High resolution near‐field fluorescence and topology images of intact neurons are reported using a cantilevered near‐field fiber optic probe. A bend is introduced into a normal geometry near‐field tip, allowing the probe to be used in a tapping‐mode arrangement, similar to tapping‐mode atomic force microscopy. Features with a full width at half‐maximum of 140 nm are observed in the near‐field fluorescence image, demonstrating the subdiffraction limit spatial resolution possible with the cantilevered near‐field probe design. Characteristics of the cantilevered tips include resonances between 30 and 60 kHz, Q factors greater than 100, and measured spring constants of 300 to 400 N/m.

Analysis of Single Bacterial Spores by Micro-Raman Spectroscopy

The spectroscopic analysis of individual living cells in their native state provides a powerful tool for the investigation of complex biological systems. Micro-Raman spectroscopy, in which confocal microscopy is combined with Raman spectroscopy, offers a promising route to achieving this, because it provides a means to study individual cells and cellular components.1–9 Here we describe the analysis of individual bacterial endospores from four species in the genus Bacillus by micro-Raman spectroscopy. Previous Raman studies on Bacillus spores resulted in spectra with strong scattering from calcium dipicolinate (CaDPA), which is the calcium chelate of dipicolinic acid (2,6-pyridinedicarboxylic acid, DPA); however, these earlier studies were conducted on concentrated samples of spores.10–14 By using micro-Raman spectroscopy, we demonstrate the ability to obtain similar information from individual spores. The Raman spectra for most spores studied were dominated by scattering from CaDPA, although Raman scattering assignable to protein bands and to phenylalanine was also observed. Approximately 4% of the spores analyzed did not exhibit Raman intensity from CaDPA, possibly due to incomplete sporulation. The results presented indicate that micro-Raman spectroscopy is a promising technique for in-

Single molecule detection and underwater fluorescence imaging with cantilevered near-field fiber optic probes

Tapping-mode near-field scanning optical microscopy (NSOM) employing a cantilevered fiber optic probe is utilized to image the fluorescence from single molecules and samples in aqueous environments. The single molecule fluorescence images demonstrate both the subdiffraction limit spatial resolution and low detection limit capabilities of the cantilevered probe design. Images taken as a function of tip oscillation drive amplitude reveal a degradation in the resolution as the amplitude is increased. With all cantilevered probes studied, however, a minimum plateau region in the resolution is reached as the drive amplitude is decreased, indicating that the tapping mode of operation does not reduce the optical resolution. Images of fluorescently doped lipid films illustrate the ability of the probe to track small height changes (<1.5 nm) in ambient and aqueous environments, while maintaining high resolution in the fluorescence image. When the tip is immersed in water (1.3 mm), the cantilevered NSOM tip resonan...

Non-DNA Methods for Biological Signatures

Publisher Summary This chapter focuses on the methods that can determine chemical or structural features of biological agent particles that are signatures of particular methods of growth and post-growth processing (often referred to as “weaponization”). The detection of these signatures in a sample of a bio-weapon (BW) agent can aid the attribution by indicating: (1) the level of sophistication of the producer, (2) the access to particular types of agent weaponization information, (3) the likelihood that the material could be or has been produced at a significant scale, (4) and by providing essential sample matching data for ascertaining a putative relationship with other samples obtained in other venues. An example of the use of biologicals in forensic science is DNA, amplied by the Polymerase Chain Reaction (PCR) technique, legally admissible in courtas evidence. DNA evidence is successfully used in the court to convict or clear people of crimes because each persons DNA is unique. High-resolution techniques are being applied to investigations; such as Environmental scanning electron microscopy (ESEM) is used for taking high-resolution images under hydrated conditions; this avoids any artifacts associated with the critical point drying process that is required under normal Scanning Electron Microscopy (SEM) operations. ESEM is also equipped with Energy Dispersive X-ray (EDX) microanalysis and Backscatter capabilities. SEM is a standard “workhorse” technique for characterizing particulate samples, found in many laboratories worldwide. It provides excellent imaging of the surfaces of agent particles and other material in a sample, and is used for identifying likely agent particles for analysis by other instruments. When combined with EDX, the elemental composition of the material in the imaged region can be determined. These techniques continue to signature libraries of correlations between analyses and growth and processing conditions of growth, it will be necessary to develop an information system which combines types of data to determine unique signatures.

Progress toward imaging biological samples with NSOM

Advancements in near-field scanning optical microscopy (NSOM) tip design as well as an interferometric feedback mechanism are presented for the common goal of imaging living biological samples under physiological conditions. The ability of a cantilevered tip to track the subtle topography changes of a fragile lipid film in an aqueous environment is demonstrated. In order to further the imaging capabilities, the probes have been chemically etched to reduce the spring constants of the tips, thereby lowering the forces imparted on the sample. An optical feedback mechanism used as an alternative to the conventional force feedback is also described. Utilizing this optical feedback mechanism, images have been obtained of fixed cells underwater. Finally, progress towards modifying the NSOM tip for chemical sensor applications is discussed in the context of eventually measuring ion fluxes through single protein channels. Together these advancements demonstrate the potential of NSOM for studying live cells.

Probing biological systems with near-field optics

The imaging characteristics of cantilevered NSOM probes operating in a tapping-mode feedback arrangement are discussed and compared to conventional tips employing the shear-force feedback method. Images form a wide range of samples are presented to demonstrate the surface tracking capabilities over both high and low topology samples, in addition to the low fluorescence detection limits possible utilizing the new tips. The results show that the cantilevered tip operating in a tapping-mode arrangement offers enhanced force imaging of the sample topology without compromising the low detection limits or high spatial resolution of the NSOM fluorescence images. The examples discussed here indicate that the new design will be particularly useful for applications involving biological samples that frequently exhibit complex surface topologies.

Application of SERS nanoparticles for intracellular pH measurements

We present an alternative approach to optical probes that will ultimately allow us to measure chemical concentrations in microenvironments within cells and tissues. This approach is based on monitoring the surface-enhanced Raman scattering (SERS) response of functionalized metal nanoparticles (50-100 nm in diameter). SERS allows for the sensitive detection of changes in the state of chemical groups attached to individual nanoparticles and small clusters. Here, we present the development of a nanoscale pH meter. The pH response of these nanoprobes is tested in a cell-free medium, measuring the pH of the solution immediately surrounding the nanoparticles. Heterogeneities in the SERS signal, which can result from the formation of small nanoparticle clusters, are characterized using SERS correlation spectroscopy and single particle/cluster SERS spectroscopy. The response of the nanoscale pH meters is tested under a wide range of conditions to approach the complex environment encountered inside living cells and to optimize probe performance.

Single molecule probes of lipid membrane dynamics

Single molecule fluorescence measurements are used to characterize the local dynamics in model lipid films of DPPC. Analysis of emission trajectory autocorrelations reveals a dependence in the single molecule emission fluctuations which is correlated with the surface pressure of the lipid monolayer. Comparison of DPPC monolayers transferred onto mica at (pi) equals 5 mN/m and (pi) equals 30 mN/m exhibit characteristic single molecule fluctuation times of 700 ms and 1.30 s, respectively. These fluctuation times are correlated with the order in the film. Comparison with recent near-field measurements of probe molecule orientation and AFM measurements of film topography, however, indicate that the lipid phase surrounding the probe molecule remains the same from (pi) equals 5 mN/m to 30 mN/m. The increase in the characteristic emission fluctuation times with increasing surface pressure, therefore, reflects a decrease in the freedom surrounding a single molecule. These fluctuations are consistent with long range concerted motions of the lipid tailgroups in the lipid film.