Christopher J. Addison

Hollow-core photonic crystal fiber-optic probes for Raman spectroscopy

We have implemented a new Raman fiber-optic probe design based on a hollow-core photonic-crystal excitation fiber surrounded by silica-core collection fibers. The photonic-crystal fiber offers low attenuation at the pump radiation wavelength, mechanical flexibility, high radiation stability, and low background noise. Because the excitation beam is transmitted through air inside the hollow-core fiber, silica Raman scattering is much reduced, improving the quality of the spectra obtained using probes of this design. Preliminary results show that the new probe design decreases the Raman background from the silica by approximately an order of magnitude compared to solid-core silica Raman probes.

Automated estimation of white Gaussian noise level in a spectrum with or without spike noise using a spectral shifting technique.

Various tasks, for example, the determination of signal-to-noise ratios, require the estimation of noise levels in a spectrum. This is generally accomplished by calculating the standard deviation of manually chosen points in a region of the spectrum that has a flat baseline and is otherwise devoid of artifacts and signal peaks. However, an automated procedure has the advantage of being faster and operator-independent. In principle, automated noise estimation in a single spectrum can be carried out by taking that spectrum, shifting a copy thereof by one channel, and subtracting the shifted spectrum from the original spectrum. This leads to an addition of independent noise and a reduction of slowly varying features such as baselines and signal peaks; hence, noise can be more readily determined from the difference spectrum. We demonstrate this technique and a spike-discrimination variant on white Gaussian noise, in the presence and absence of spike noise, and show that highly accurate results can be obtained on a series of simulated Raman spectra and consistent results obtained on real-world Raman spectra. Furthermore, the method can be easily adapted to accommodate heteroscedastic noise.

Base Stacking Configuration is a Major Determinant of Excited State Dynamics in A. T DNA and LNA

Base stacking plays an important role in excited state dynamics in polynucleotides. However, it is poorly un- derstood how stacking geometries influence the formation of and relaxation from excites states. Natural poly(dA)·poly(dT) adopts a B-form structure with extensive geometrical overlap between adjacent stacked adenines while the synthetic, locked ribose analogue (LNA), adopts the A-form structure where such overlap between adjacent adenines is reduced. We have used pump-probe transient absorption measurements on DNA and LNA, with excitation at 260 nm and absorption monitored at 440 and 260 nm, to examine the differences in excited state dynamics in B- and A-form con- formations. We observed slow decay times, both early and late stage, from the excited states of B-form and fast decay times from the excited states of analogous homopolymeric A-form structures. Within similar conformations, relaxation times are dependent on the number of stacked adenines as determined by either chain length or sequence. An increase in excited state lifetimes with increase in the number of stacked adenines shows that these excited states can be delocalized over several bases. Thus excited state lifetimes are highly dependent on how the bases are stacked. We conclude from our results that, for identical sequences, conformations that exhibit a high degree of adenine base overlap favor initial coop- erative excitation as well as subsequent evolution to delocalized excited states, but hinder the formation of out-of-plane geometries required for fast relaxation to the electronic ground state thus prolonging excited state lifetimes.

Exploring the potential of Raman and resonance Raman spectroscopy for quantitative analysis of duplex DNA

Advances in DNA microarray fabrication technologies, expanding probe libraries, and new bioinformatics methods and resources have firmly established array-based techniques as mainstream bioanalytical tools and the application space is proliferating rapidly. However, the capability of these tools to yield truly quantitative information remains limited, primarily due to problems inherent to the use of fluorescence imaging for reading the hybridized arrays. The obvious advantages of fluorescence are the unrivaled sensitivity and simplicity of the instrumentation. There are disadvantages of this approach, however, such as difficulties in achieving optimal labeling of targets and reproducible signals (due to quenching, resonance energy transfer, photobleaching effects, etc.) that undermine precision. We are exploring alternative approaches, based mainly on Raman and resonance Raman spectroscopy, that in principle permit direct analysis of structural differences between hybridized and unhybridized probes, thereby eliminating the need for labeling the target analytes. We report here on the status of efforts to evaluate the potential of these methods based on a combination of measured data and simulated experiments involving short (12-mer) ssDNA oligomer probes with varying degrees of hybridized target DNA. Preliminary results suggest that it may be possible to determine the fraction of duplex probes within a single register on a DNA microarray from 100% down to 10% (or possibly less) with a precision of ±2 5%. Details of the methods used, their implementation, and their potential advantages and limitations are presented, along with discussion of the utility of using 2DCOS methods to emphasize small spectral changes sensitive to interstrand H bonding, backbone flexibility, hypochromicity due to base-stacking in duplex structures and solvation effects.