Richard W. Bormett

UV Resonance Raman Spectroscopy Using a New cw Laser Source: Convenience and Experimental Simplicity

A new laser has been developed which generates hundreds of milliWatts of cw UV power below 260 nm. The laser consists of a small-frame Ar+-ion laser which is intracavity doubled with the use of BBO nonlinear optical crystals. More than 300 mW are available at 244 and 257 nm, while 180, 100, and 30 mW are available at 248, 238, and 228.9 nm, respectively. This laser is an ideal source for UV Raman spectroscopy since it avoids the nonlinear and saturation problems common with the typical pulsed laser excitation sources. It also minimizes thermal sample degradation. We demonstrate the increased spectral signal-to-noise ratios possible due to the ability to focus the cw laser into a small-volume element that can be efficiently imaged into the spectrometer. We demonstrate the ability of this laser to excite Raman spectra of solid samples such as coal-liquid residuals, and point out the utility of the 228.9-nm line for studying aromatic amino acids in proteins. We also demonstrate the ability to selectively study pyrene intercalated into calf thymus DNA.

Ultraviolet Raman spectroscopy characterizes chemical vapor deposition diamond film growth and oxidation

The Raman spectra of diamond and chemical‐vapor‐deposition (CVD) diamond films in the UV have been excited within the diamond band gap at 228.9 nm for the first time. The lack of fluorescence in the UV‐excited Raman spectrum of diamond and CVD diamond films allows Raman spectroscopy to monitor the carbon‐hydrogen (C‐H) stretching vibrations of the nondiamond components of the CVD film as well as the third‐order phonon bands of diamond. The relative intensity of the C‐H stretching bands at ∼2930 cm−1 to the diamond first‐order phonon band at 1332 cm−1 is proportional to the atomic fraction of covalently bound hydrogen in the CVD diamond film. The third‐order phonon band intensity and frequency maxima are very sensitive to the size of the diamond crystallite. Its intensity decreases, and the maximum shifts to lower frequency as the size of the diamond crystallite decreases. It is shown here that UV Raman diamond measurements have significantly greater information content than visible Raman measurements.

UV RAMAN MICROSPECTROSCOPY: SPECTRAL AND SPATIAL SELECTIVITY WITH SENSITIVITY AND SIMPLICITY

The high sensitivity, selectivity, spatial resolution, and ease of operation of UV Raman microspectr oscopy is demonstrated with the use of a new highly efficient UV Raman microspectrometer with excitation at 244 nm. Single spectrograph dispersion combined with special new filters for the rejection of Rayleigh scattering improves the throughput efficiency by a factor of approximately 4 in comparison to a triple-stage spectrograph. The instrument has a spatial resolution of approximately 3 μm × 9 μm in the lateral (X–Y) plane, and 10 μm or less in the axial (Z) plane. UV resonance Raman spectra of nucleic acids are selectively excited from spatially resolved areas of a single paramecium by using low continuous-wave (cw) excitation powers and short accumulation times to minimize sample damage. High signal-to-noise Raman spectra are excited from spatially resolved areas of chemical-vapor-deposited (CVD) diamond films. We demonstrate, for the first time, the ability to probe the spatial distribution of the nondiamond carbon impurities in CVD diamond films. The amorphous carbon band at ∼ 1553 cm−1 is resolved from the normally broad ∼ 1600-cm−1 nondiamond carbon band.

Applications of a New 206.5-nm Continuous-Wave Laser Source: UV Raman Determination of Protein Secondary Structure and CVD Diamond Material Properties

We demonstrate the utility of a new 206.5-nm continuous-wave UV laser excitation source for spectroscopic studies of proteins and CVD diamond. Excitation at 206.5 nm is obtained by intracavity frequency doubling the 413-nm line of a krypton-ion laser. We use this excitation to excite resonance Raman spectra within the π → π amide transition of the protein peptide backbone. The 206.5-nm excitation resonance enhances the protein amide vibrational modes. We use these high signal-to-noise spectral data to determine protein secondary structure. We also demonstrate the utility of this source to excite CVD and gem-quality diamond within its electronic bandgap. The diamond Raman spectra have very high signal-to-noise ratios and show no interfering broad-band luminescence. Excitation within the diamond bandgap also gives rise to narrow photoluminescence peaks from diamond defects. These features have previously been observed only by cathodoluminescence measurements. This new continuous-wave UV source is superior to the previous pulsed sources, because it avoids nonlinear optical phenomena and thermal sample damage; Photoluminescence.

2-D LIGHT DIFFRACTION FROM CCD AND INTENSIFIED RETICON MULTICHANNEL DETECTORS CAUSES SPECTROMETER STRAY LIGHT PROBLEMS

Intensified diode arrays and charge-coupled detectors (CCD) which are used as multichannel detectors for spectroscopy exhibit strong 2-D diffraction of light due to the micro-channel plate intensifier and the CCD surface microelectronic structures. The strong 2-D diffraction of light by the intensified diode arrays shows hexagonal symmetry due to the hexagonal packing of the hollow glass fibers of the micro-channel plate intensifier. The 2-D diffraction of light from the CCD detectors shows square symmetry due to the almost square symmetry of the individual surface microelectronic structures. Light incident on the detector surfaces is diffracted into numerous angles which depend upon the incident angle and the light wavelength. This diffracted light can be redispersed and/ or reflected and scattered by optical elements inside the spectrometer. This diffracted light can then contribute to spectrometer diffuse stray light or it can be directly reimaged onto the detector to cause spectral artifacts. Backthinned CCD detectors do not show 2-D light diffraction and thus avoid these 2-D diffraction stray light limitations.

Vibrational circular dichroism measurements of ligand vibrations in haem and non-haem metalloenzymes

We have measured the vibrational circular dichroism (VCD) spectra of the stretching vibrations of azide and cyanide ligated to the Fe3+ atoms of haemoglobin (Hb) and myoglobin (Mb). The antisymmetric azide-stretch of the low-spin haems have an anomalously large g-value of ca. -1 x 10(-3). In contrast, CN- has a g-value of ca. +2.4 x 10(-3). We also show, for the first time, that a significant VCD occurs for the azide ligand antisymmetric stretches of non-haem proteins; we measure a g-value of ca. -1 x 10(-4) for azide bound to haemerythrin. We have examined the mechanism of the VCD phenomenon by: (1) reconstituting Mb with haems substituted such that they insert differently in the haem pocket; (2) replacing the Fe3+ with Mn3+; (3) examining proteins where replacements occur for E-7 His and E-11 Val distal amino acids close to the haem and (4) examining an Mb mutant where the proximal F-8 His is replaced by Gly, and where an imidazole ligand inserts into the resulting crevice and binds to the haem in a way similar to that of the proximal histidine in the native protein. The VCD anisotropy appears insensitive to the haem substituent replacements used in this study. Exchange of the E-7 distal His or the E-11 Val has a dramatic effect on the g-value. Exchange of the F-8 proximal His reverses the sign of the g-value for the azide complex, but not for the cyanide complex. The work to date indicates that VCD has the potential to become a sensitive technique for examining the structure of metalloenzymes. Work is needed to determine the mechanism giving rise to the large g-values and to correlate the VCD spectrum with the metalloenzyme structure at the active site.

Convenient microsampling system for UV resonance Raman spectroscopy

We report here the development of a new microsampling device for UV Raman measurements which permits the use of sample volumes as small as 50 μL and which permits recirculation of the sample to minimize sample heating and the contribution of transient species. UV Raman studies with pulsed laser sources (typically ~ 10 ns duration) can cause transient heating during the laser pulse. Unless the sample is exchanged between pulses, the Raman measurements will monitor previously heated sample volumes which may contain thermally or photochemically generated transient species. Given the ~ 10 ms intervals between laser pulses, we may observe photochemically generated transient species with decay times longer than ~ 1 ms. In addition, the long thermal diffusion times which exist for typical samples will result in the significant heating of a static illuminated sample volume.