Measurement of some small-molecule and peptide neurotransmitters in-vitro using a fiber-optic probe with pulsed ultraviolet resonance Raman spectroscopy.

Many techniques have been developed to investigate the chemistry associated with brain activity. These techniques generally fall into two categories: fast techniques with species-limited sensitivity; and generally slower techniques with broader species sensitivity. Therefore, a need exists for a fast, minimally invasive technique that is sensitive to a wide array of biologically relevant compounds in order to measure chemical brain events in real time. The work presented here describes the development of a novel spectroscopic neurotransmitter probe for the rapid and simultaneous detection of a variety of neurotransmitters. A fiber-optic-linked Raman and tunable ultraviolet resonance Raman system was assembled with custom designed optical fiber probes. Using this system, the ultraviolet resonance Raman spectra of some small-molecule and peptide neurotransmitters were measured in-vitro with a fiber-optic probe and are reported here for the first time. The probe has furthermore been used to measure neurotransmitter secretions obtained from depolarized rat pheochromocytoma (PC12) cells. These results demonstrate the general utility of this approach which, due to the fiber-optic implementation, could potentially also be applied to in-vivo neurotransmitter determinations.

Fiber-optic probes with improved excitation and collection efficiency for deep-UV Raman and resonance Raman spectroscopy.

The ability of ultraviolet resonance Raman spectroscopy (UVRRS) to determine structural, environmental, and analytical information concerning low-concentration aqueous biomolecules makes it a powerful bioanalytical and biophysical technique. Unfortunately, its utility has been limited by experimental requirements that preclude in situ or in vivo studies in most cases. We have developed the first high-performance fiber-optic probes suitable for long-term use in pulsed UVRRS applications in the deep- UV (DUV, 205-250 nm). The probes incorporate recently developed improved ultraviolet (IUV) fibers that do not exhibit the rapid solarization and throughput decay that previously hampered the use of optical fibers for delivering pulsed, DUV light. A novel 90 degrees mirrored collection geometry is used to overcome the inner-filtering effects that plague flush-probe geometries. The IUV fibers are characterized with respect to their efficacy at transmitting pulsed, DUV laser light, and prototype probes are used to obtain pulsed UVRRS data of aromatic amino acids, proteins, and hormones at low concentrations with 205-240-nm pulsed excitation. Efficient probe geometries and fabrication methods are presented. The performance of the probes in examining resonance-enhanced Raman signals from absorbing chromophores is investigated, and the optimal excitation wavelength is shown to be significantly red-shifted from the maximum of the resonance Raman enhancement profile. Generally applicable procedures for determining optimal experimental conditions are also introduced.

Rational design of fiber-optic probes for visible and pulsed-ultraviolet resonance Raman spectroscopy.

We investigated the performance of fiber-optic resonance Raman probes with a series of experiments to determine the working curves of such probes using model analytes and to investigate the effects of absorbing media. A computer model designed to simulate these experiments is presented, and numerical results are found to be in agreement with the experimental data. Design considerations resulting from these studies are discussed, and novel designs for overcoming problems of coupling efficiency, damage threshold, and sensitivity in absorbing samples are presented. These findings are applied to the design of fiber-optic probes for ultraviolet resonance Raman spectroscopy involving nanosecond pulsed-ultraviolet excitation (225 and 266 nm). These probes have been used to collect what is, to our knowledge, the first reported fiber-optic-linked ultraviolet resonance Raman spectra of tryptophan and DNA.

Artificial neural network and classical least-squares methods for neurotransmitter mixture analysis.

Identification of individual components in biological mixtures can be a difficult problem regardless of the analytical method employed. In this work, Raman spectroscopy was chosen as a prototype analytical method due to its inherent versatility and applicability to aqueous media, making it useful for the study of biological samples. Artificial neural networks (ANNs) and the classical least-squares (CLS) method were used to identify and quantify the Raman spectra of the small-molecule neurotransmitters and mixtures of such molecules. The transfer functions used by a network, as well as the architecture of a network, played an important role in the ability of the network to identify the Raman spectra of individual neurotransmitters and the Raman spectra of neurotransmitter mixtures. Specifically, networks using sigmoid and hyperbolic tangent transfer functions generalized better from the mixtures in the training data set to those in the testing data sets than networks using sine functions. Networks with connections that permit the local processing of inputs generally performed better than other networks on all the testing data sets. and better than the CLS method of curve fitting, on novel spectra of some neurotransmitters. The CLS method was found to perform well on noisy, shifted, and difference spectra.