Development of a combined confocal and scanning near‐field Raman microscope for deep UV laser excitation

Sands, H. S.; Demangeot, F.; Bonera, Emiliano; Webster, S.; Bennett, Robert D.; Hayward, Ian; Marchi, Florence; Smith, D. A.; Batchelder, D. N.

doi:10.1002/jrs.909

Cited by 27 publications

(15 citation statements)

References 41 publications

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“…The UV system was adapted for operation with a 244 nm laser as described elsewhere. 9,11 A Raman spectrum of a silicon wafer was recorded with each Raman microscope (diamond spectrum in case of the UV microscope) prior to the experiments. The silicon and diamond Raman bandwidths were used to estimate the spectral resolution of the instrument; the results, and laser power at the sample, are shown in Table 1.…”

Section: Methodsmentioning

confidence: 99%

Dependence of the Raman spectra of drug substances upon laser excitation wavelength

Thorley

Baldwin

Lee

et al. 2006

J Raman Spectroscopy

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The laser excitation wavelength is an important parameter in obtaining definitive Raman spectra from pharmaceutical drug substances. This paper investigates the effect of changing wavelength on the Raman spectra of four different drug substances: paracetamol, paroxetine and ranitidine (polymorphic forms I and II). Excitation wavelengths in the range 244 nm to 785 nm have been used. The fluorescence intensities in the Raman spectra were found to be negligible at the two extreme laser wavelengths but tended to interfere in the visible range. Resonance Raman enhancement was observed with 244 nm excitation but there was evidence for sample degradation. Furthermore, the site selective nature of the resonance enhancement reduced the differences between the Raman spectra of the two ranitidine polymorphs.

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Section: Methodsmentioning

confidence: 99%

Dependence of the Raman spectra of drug substances upon laser excitation wavelength

Thorley

Baldwin

Lee

et al. 2006

J Raman Spectroscopy

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“…UVRR spectra were obtained with a Renishaw Raman System 1000 spectrometer described elsewhere (15), adapted for use at 229 and 244 nm using approximately 0.3 mW at the sample generated by an (36) and Raster3D (37) from the coordinates of the crystal structure of Im7 (PDB entry 1AYI) (1). (B) Fluorescence emission spectra of the folded states of wild-type Im7* (black), L53AI54A (green), H3G6 (pink), and YY (light blue) relative to the denatured states of each protein in 8 M urea, and the 8 M urea denatured state of wild-type Im7* (dashed black).…”

Section: Methodsmentioning

confidence: 99%

Ultraviolet Resonance Raman Studies Reveal the Environment of Tryptophan and Tyrosine Residues in the Native and Partially Folded States of the E Colicin-Binding Immunity Protein Im7

et al. 2005

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Understanding the nature of partially folded proteins is a challenging task that is best accomplished when several techniques are applied in combination. Here we present ultraviolet resonance Raman (UVRR) spectroscopy studies of the E colicin-binding immunity proteins, Im7* and Im9*, together with a series of variants of Im7* that are designed to trap a partially folded state at equilibrium. We show that the environments of the tryptophan and tyrosine residues in native wild-type Im7* and Im9* are indistinguishable, in contrast with models for their structures based on X-ray and NMR methods. In addition, we show that there is a general increase in the hydrophobicity in the environment of Trp75 in all of the variants compared with wild-type Im7*. These data suggest that a significant rearrangement of the tryptophan pocket occurs in the variants, which, together with an overall decrease in solvent accessibility of Trp75 as judged by time-resolved fluorescence lifetime measurements and fluorescence quenching experiments, rationalize the unusual fluorescence properties of the variants reported previously. The data highlight the power of UVRR in analyzing the structural properties of different conformational states of the same protein and reveal new information about the structural rearrangements occurring during Im7* folding, not possible using other spectroscopic methods alone. Finally, we describe a previously unreported dependence of the tryptophan Fermi doublet on excitation wavelength in the ultraviolet region revealed by these protein spectra. We corroborated this observation using tryptophan-containing model compounds and conclude that the conventional interpretation of this UVRR feature at these wavelengths is unreliable.The biological role of the E colicin-binding immunity proteins Im2, Im7, Im8, and Im9 is to protect the host cell from the toxic endonuclease activity of a coexpressed cognate colicin, by binding to an exosite in the DNase domain, inhibiting access of the substrate DNA to the active site of the colicin by steric hindrance and electrostatic repulsion (1, 2). The small size of the immunity proteins (85-87 amino acid residues), their high level of sequence identity (approximately 60%), the single conserved tryptophan, and their lack of disulfide bonds, prosthetic groups, and cis-prolines in the native state make the immunity protein family ideal for protein folding studies. Despite the high degree of similarity between Im7 and Im9 in both sequence and structure, these proteins fold by mechanisms with different levels of kinetic complexity at neutral pH, suggesting that local sequence variations, rather than topology or global stability, are responsible for the observed folding kinetics of these proteins (3-7). Im7* 1 and Im9* (hexahistidinetagged analogues of Im7 and Im9) contain four helices ( Figure 1A), each of which displays polar side chains on the surface of the protein, while the majority of hydrophobic side chains are buried, forming a well-packed hydrophobic core. Im7* and Im...

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“…Raman signals excited by UV light are much stronger for some materials such as carbon films due to resonance effect. 6 The drawbacks such as cost, sensitivity of detector and difficult system adjustment make UV Raman less popular as compared with visible and infrared Raman. The infrared Raman is a good choice for suppressing fluorescence.…”

Section: Introductionmentioning

confidence: 99%

Two-dimensional surface characterization of laser-deposited carbon films using Raman scattering

Líu

2006

Laser-Based Micropackaging

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We report an apparatus designed to characterize two-dimensional (2D) surfaces of carbon films based on the principle of inelastic light scattering (Raman scattering). The design and construction details are presented. The system with a backscattering configuration, is constructed using a high power argon ion laser with a wavelength of 514.5 nm, an XYZ motorized stage with a step resolution of 3.175 µm, a microscope objective lens, a confocal spatial filter and a holographic notch filter, to achieve extremely low crosstalk and maximum resolution in spectroscopy. The radial resolution for film surface is much enhanced by confocal spatial filter due to its stray light suppression capability. A large depth of sampling field is achieved using an objective lens with a middle NA of 0.55 and a long working distance of 8 mm, thus the requirement of using auto-focusing can be avoided. A specific algorithm is designed to decide the film boundaries as well as the outline of surface structures from pre-defined spectral windows. Control software on Labview TM platform has been developed for controlling movement of the sample stage, spectral acquisition and data visualization. Single-walled carbon nanotubes (SWCNTs) and patterned silicon were used to evaluate the sensitivity, 1D profile and 2D mapping functionality of the designed system. Diamond-like amorphous carbon (DLC) films prepared by pulsed-laser deposition (PLD) were studied using the developed instrument. The results from this approach are compared with those using general scanning tunneling microscope (STM). This comparable low-cost system with high performance is suitable to characterize semiconductors and other materials both for industrial applications and academic research.

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Development of a combined confocal and scanning near‐field Raman microscope for deep UV laser excitation

Cited by 27 publications

References 41 publications

Dependence of the Raman spectra of drug substances upon laser excitation wavelength

Dependence of the Raman spectra of drug substances upon laser excitation wavelength

Ultraviolet Resonance Raman Studies Reveal the Environment of Tryptophan and Tyrosine Residues in the Native and Partially Folded States of the E Colicin-Binding Immunity Protein Im7

Two-dimensional surface characterization of laser-deposited carbon films using Raman scattering

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