Kevin L. Davis

Measurement of Spatial Resolution and Sensitivity in Transmission and Backscattering Raman Spectroscopy of Opaque Samples: Impact on Pharmaceutical Quality Control and Raman Tomography:

A practical methodology is described that allows measurement of spatial resolution and sensitivity of Raman spectroscopy in backscatter and transmission modes under conditions where photon migration dominates, i.e., with turbid or opaque samples. For the first time under such conditions the width and intensity of the point spread function (PSF) has been accurately measured as a function of sample thickness and depth below the surface. In transmission mode, the lateral resolution for objects in the bulk degraded linearly with sample thickness, but the resolution was much better for objects near either surface, being determined by the diameter of the probe beam and collection aperture irrespective of sample thickness. In other words, buried objects appear to be larger than ones near either surface. The absolute transmitted signal decreased significantly with sample thickness, but objects in the bulk yielded higher signals than those at either surface. In transmission, materials are sampled preferentially in the bulk, which has ramifications for quantitative analysis. In backscattering mode, objects near the probed surface were detected much more effectively than in the bulk, and the resolution worsened linearly with depth below the surface. These results are highly relevant in circumstances in which one is trying to detect or image buried objects in opaque media, for example Raman tomography of biological tissues or compositional and structural analysis of pharmaceutical tablets. Finally, the observations were in good agreement with Monte Carlo simulations and, provided one is in the diffusion regime, were insensitive to the choice of transport length, which shows that a simple model can be used to predict instrument performance for a given excitation and collection geometry.

DENSITY MAPPING IN POLY-ETHYLENE TEREPHTHALATE) USING A FIBER-COUPLED RAMAN MICROPROBE AND PARTIAL LEAST-SQUARES CALIBRATION

Partial least-squares (PLS) analysis has been used to calibrate Raman microprobe spectra of poly(ethylene terephthalate) films in terms of density, in order to give insight into changes in crystallinity through the film thickness. The microprobe utilizes a static multiplexed holographic grating to obtain the entire Raman spectrum (-1600-4000 cm-1) in a “single-shot” at ∼ 5-cm−1 resolution. Because there are no moving parts, frequency registration and repeatability are excellent and ideally suited for multivariate calibration. In addition, the high spectral throughput allows the whole spectrum to be collected in a few seconds with high signal-to-noise ratio. With this equipment, cross-validated calibration precisions as low as - 0.0021 g cm−3 were achieved. In this work we considered two ways of removing fluorescence backgrounds prior to carrying out multivariate calibration. The first involved manually fitting a baseline using a polynomial curve and subtracting it. The second approach simply takes the second derivative of the spectrum to attenuate the low-frequency components (i.e., the curved baseline). It was found that either pretreatment gave good calibration precision provided that the resultant spectra were intensity-normalized to correct for variations in laser power, sample alignment, and so on. Surprisingly, it was found that the best precision was obtained by grouping the spectral resolution elements into blocks of eight data points, thereby improving the signal-to-noise but effectively degrading the spectral resolution by a factor of three. This was especially important for the derivative spectra. Alternatively, Savitsky-Golay smoothing of the second derivative data was applied to the same effect but also at the expense of degrading spectral resolution. The implication of this work is that instruments intended for multivariate calibration applications could perhaps be designed to work at rather lower spectral resolutions (but higher signal-to-noise) than might otherwise be considered.

Surface-enhanced resonance Raman spectroscopy of iron-dopamine complexes

Abstract Surface-enhanced resonance Raman spectroscopy (SERRS) at silver colloids is used to detect the catecholamines, 3-hydroxytyramine (dopamine) and 3,4-dihydroxyphenylacetic acid (DOPAC), in a modified Ringers solution. Catecholamines form very strong complexes with iron(III) in solution ( K f > 10 40 ) and exhibit a broad ligand-to-metal charge-transfer (LMCT) absorption in the visible (∼ 500 nm). Resonance enhancement is achieved by excitation at 532 nm from a frequency doubled Nd:YAG laser with high quality spectra attainable in 1 s. Maximum SERRS signal is observed when basic buffer is added to a dopamine sample containing 50 × 10 −6 M ferric ion. Dopamine concentrations in the nanomolar (resting level) range are obtained using this technique.

SERS Microscopy: Laser Illumination Effects

Hadamard transform SERS microscopy of pyridine at Ag electrodes with focused 514.5-nm, 532.0-nm, and 632.8-nm excitation is reported. It is shown that focused 514.5-nm and 532.0-nm laser excitation produces photochemical or photothermal damage at roughened Ag electrodes. Photo-damage limits the spatial resolution of SERS microprobe and raster-scanned point imaging techniques which use focused green excitation at Ag electrodes.

Surface enhanced Raman spectroscopy of neurotransmitters

The surface‐enhanced Raman spectra (SERS) of neurotransmitters in biological matrices and synthetic solutions are described. The effects of protein adsorption on cathecholamine SERS intensity are discussed. Techniques for obtaining dopamine SERS spectra in cerebrospinal fluid and rat brain dialysate are demonstrated. Preliminary SERS of histamine and tel‐methylhistamine are presented.

Surface-enhanced Raman probes of neurotransmitters

SERS of catecholamines and histamines is discussed. The SERS of histamines is partially assigned. The use of cellulose acetate electrodes for SERS in matrices with a high protein burden is described.