Helium-cadmium Laser Research Articles

Laser-induced Propagation and Destruction of Amyloid β Fibrils

The amyloid deposition of amyloid beta (Abeta) peptides is a critical pathological event in Alzheimer disease (AD). Preventing the formation of amyloid deposits and removing preformed fibrils in tissues are important therapeutic strategies against AD. Previously, we reported the destruction of amyloid fibrils of beta(2)-microglobulin K3 fragments by laser irradiation coupled with the binding of amyloid-specific thioflavin T. Here, we studied the effects of a laser beam on Abeta fibrils. As was the case for K3 fibrils, extensive irradiation destroyed the preformed Abeta fibrils. However, irradiation during spontaneous fibril formation resulted in only the partial destruction of growing fibrils and a subsequent explosive propagation of fibrils. The explosive propagation was caused by an increase in the number of active ends due to breakage. The results not only reveal a case of fragmentation-induced propagation of fibrils but also provide insights into therapeutic strategies for AD.

Optimization of synthesis conditions and composition of blue phosphors [(B2O3)0.5(Al2O3)0.5] · 2SiO2: Eu2+

It is found that [(B2O3)0.5(Al2O3)0.5] · 2SiO2: Eu2+ systems with different concentrations of europium exhibit intense photoluminescence in the blue-violet range. When the system is excited by a 325-nm helium-cadmium laser, the spectra of blue phosphors exhibit a maximum in the interval 419–439 nm, while excitation by a pulsed nitrogen laser with a wavelength of 337 nm causes luminescence between 429 and 461 nm. Phosphor with the composition [(B2O3)0.5(Al2O3)0.5] · 2SiO2: Eu (3 mol %) (x = 0.151, y = 0.062) excited by the radiation of a helium-cadmium laser and phosphor of the same composition having been subjected to additional vacuum annealing and excited by the radiation of a nitrogen laser (x = 0.145, y = 0.084) are found to be optimal to provide primary blue color in the EBU and NTSC TV standards. Phosphors of the composition [(B2O3)0.5(Al2O3)0.5] · 2SiO2: Eu2+ (7 mol %) offer the highest luminescence intensity both before and after vacuum annealing.

Optical and Micro-structural Properties of GaN Nanowires by Ammoniating Ga2O3/Nb Films

Single-crystalline GaN nanowires have been synthesized on Si(111) substrates by magnetron sputtering through ammoniating the Ga 2 O 3 /Nb films at 900 °C in a quartz tube. The as-prepared nanowires are confirmed as single crystalline GaN with wurtzite structure by X-ray diffraction (XRD), selected-area electron diffraction (SAED) and high-resolution transmission electron microscopy (HRTEM). Transmission electron microscopy (TEM) shows that the GaN nanowires are straight and smooth, and possess the diameters of about 50 nm and lengths up to several microns. When excited by 325 nm helium-cadmium (He-Cd) laser light at room temperature, the GaN nanowires only have a strong ultraviolet luminescence peak located at 367 nm, owing to GaN band-edge emission. Finally, the growth mechanism of GaN nanowires is discussed briefly.

Photoluminescence degradation in GaN induced by light enhanced surface oxidation

The exponential degradation of the photoluminescence (PL) intensity at the near-band-gap was observed in heavily doped or low-quality GaN with pristine surface under continuous helium-cadmium laser excitation. In doped GaN samples, the degradation speed increased with doping concentration. The oxidation of the surface with laser irradiation was confirmed by x-ray photoemission spectroscopy measurements. The oxidation process introduced many oxygen impurities and made an increase of the surface energy band bending implied by the shift of Ga 3d binding energy. The reason for PL degradation may lie in that these defect states act as nonradiative centers and/or the increase of the surface barrier height reduces the probability of radiative recombination.

Substrate-specific Interactions with the Heme-bound Oxygen Molecule of Nitric-oxide Synthase

We report the characterization by resonance Raman spectroscopy of the oxygenated complex (Fe(II)O(2)) of nitric-oxide synthases of Staphylococcus aureus (saNOS) and Bacillus subtilis (bsNOS) saturated with N(omega)-hydroxy-l-arginine. The frequencies of the nu(Fe-O) and nu(O-O) modes were 530 and 1135 cm(-), respectively, in both the presence and absence of tetrahydrobiopterin. On the basis of a comparison of these frequencies with those of saNOS and bsNOS saturated with l-arginine (nu(Fe-O) at 517 cm(-1) and nu(O-O) at 1123 cm(-1)) and those of substrate-free saNOS (nu(Fe-O) at 517 and nu(O-O) at 1135 cm(-1)) (Chartier, F. J. M., Blais, S. P., and Couture, M. (2006) J. Biol. Chem. 281, 9953-9962), we propose two models that account for the frequency shift of nu(Fe-O) (but not nu(O-O)) upon N(omega)-hydroxy-l-arginine binding as well as the frequency shift of nu(O-O) (but not nu(Fe-O)) upon l-arginine binding. The implications of these substrate-specific interactions with respect to catalysis by NOSs are discussed.

Sub-100 nm photolithography using TE-polarized waves in transparent nanostructures

The optical near field and its polarization anisotropy in transparent nanostructures were studied by polarization near-field optical microscopy. From experimental results and finite-difference time-domain calculations, we conclude that localized optical near fields exist at topographically higher regions of nanostructures under the TE-polarization condition. Optical near fields with a feature size smaller than 100 nm are applied for contact photomask lithography. We demonstrate photolithographic patterns with 80 nm width by using a 442 nm helium cadmium laser.

Real-time and Single Fibril Observation of the Formation of Amyloid β Spherulitic Structures

In Alzheimer disease, amyloid beta, a 39-43-residue peptide produced by cleavage from a large amyloid precursor protein, undergoes conformational change to form amyloid fibrils and deposits as senile amyloid plaques in the extracellular cerebral cortices of the brain. However, the mechanism of how the intrinsically linear amyloid fibrils form spherical senile plaques is unknown. With total internal reflection fluorescence microscopy combined with the use of thioflavin T, an amyloid-specific fluorescence dye, we succeeded in observing the formation of the senile plaque-like spherulitic structures with diameters of around 15 microm on the chemically modified quartz surface. Real-time observation at a single fibrillar level revealed that, in the absence of tight contact with the surface, the cooperative and radial growth of amyloid fibrils from the core leads to a huge spherulitic structure. The results suggest the underlying physicochemical mechanism of senile plaque formation, essential for obtaining insight into prevention of Alzheimer disease.

Protein Kinase C δ (δPKC)-Annexin V Interaction: A REQUIRED STEP IN δPKC TRANSLOCATION AND FUNCTION

Protein Kinase C δ (δPKC)-Annexin V Interaction: A REQUIRED STEP IN δPKC TRANSLOCATION AND FUNCTION

Safety and Efficacy of Ultraviolet-A Light-Activated Gene Transduction for Gene Therapy of Articular Cartilage Defects

Gene therapies for articular cartilage defects are limited by the absence of an in vivo delivery system that can mediate site-specific transduction restricted to within the margins of the defect during routine arthroscopy. We have proposed the use of ultraviolet light to stimulate gene expression following infection by recombinant adeno-associated virus (rAAV). However, research has demonstrated that short-wavelength ultraviolet light (ultraviolet C), while effective, is neither safe nor practical for this purpose. We evaluated the safety and efficacy of long-wavelength ultraviolet light (ultraviolet A) from a laser to induce light-activated gene transduction in articular chondrocytes in vitro and in vivo. The effects of ultraviolet A from a 325-nm helium-cadmium laser, delivered through a fiberoptic cable, on cytotoxicity, mutagenesis, intracellular reactive oxygen species, and light-activated gene transduction of human articular chondrocytes were evaluated in dose-response experiments of primary cultures. Cytotoxicity was determined by trypan blue exclusion. The presence of pyrimidine dimers in purified genomic DNA was determined by enzyme-linked immunosorbent assays. Intracellular reactive oxygen species levels were determined by flow cytometry at one hour and twenty-four hours. In vitro light-activated gene transduction with rAAV vectors expressing the green fluorescent protein (eGFP) or beta-galactosidase (LacZ) was determined by fluorescence microscopy and bioluminescence assays, respectively. In vivo light-activated gene transduction was quantified by stereotactic immunohistochemistry for beta-galactosidase in rabbit articular cartilage defects in the patellar groove that had been irradiated with +/-6000 J/m2 of ultraviolet A one week after direct injection of 10(7) transducing units of rAAV-eGFP. Ultraviolet A failed to induce significant cytotoxicity at all fluencies below 6000 J/m2. Dose-dependent cytotoxicity was observed at greater fluencies. In contrast to ultraviolet C, which induced significant (p < 0.05) pyrimidine dimer formation at all fluencies in a dose-dependent manner, ultraviolet A failed to induce DNA modifications. Conversely, ultraviolet C proved to be a poor inducer of intracellular reactive oxygen species, while ultraviolet A immediately induced high levels of intracellular reactive oxygen species, which were completely resolved twenty-four hours later. Ultraviolet A demonstrated significant light-activated gene transduction effects in vitro, which were dose-dependent (p < 0.05). In vivo, ultraviolet A mediated a tenfold increase in transduction in which 40.8% of the superficial chondrocytes adjacent to the defect stained positive for green fluorescent protein compared with 5.2% in the knees treated with no ultraviolet A (p < 0.006). These results provide what we believe is the first formal demonstration of an agent that can induce rAAV transduction in the complete absence of cytotoxicity and DNA modification. They also suggest that the mechanism by which long-wavelength ultraviolet light mediates site-specific gene expression is by means of the induction of intracellular reactive oxygen species. Finally, laser-derived ultraviolet A can be readily transferred through a fiberoptic cable to mediate light-activated gene transduction in vivo.

Capillary electrophoresis combined with microdialysis in the human spinal cord: a new tool for monitoring rapid peroperative changes in amino acid neurotransmitters within the dorsal horn.

A method originally developed for the separation of the three neurotransmitters gamma-aminobutyric acid (GABA), glutamate (Glu) and L-aspartate (L-Asp) in microdialysis samples from rat brain (Sauvinet et al., Electrophoresis 2003, 24, 3187-3196) was applied to human spinal dialysates obtained during peroperative microdialysis from patients undergoing surgery against chronic pain. Molecules were tagged on their primary amine function with the fluorogene agent, naphthalene-2,3-dicarboxaldehyde (NDA), and, after separation by capillary electrophoresis (CE, 75 mmol/L borate buffer, pH 9.2, containing 70 mmol/L sodium dodecyl sulfate and 10 mmol/L hydroxypropyl-beta-cyclodextrin, + 25 kV voltage), were detected by laser-induced fluorescence detection (LIFD) using a 442 nm helium-cadmium laser. The complete method, including microdialysis sampling and analysis by CE-LIFD, has been validated for the analysis of human spinal microdialysates. The analytical detection limits were 1, 3.7 and 17 nmol/L for GABA, Glu and L-Asp respectively. This method allows an accurate measurement of the three amino acid neurotransmitters during an in vivo monitoring performed as rapidly as every minute in the human spinal dorsal horn. In addition, the effect of a brief peroperative electrical stimulation of the dorsal rootlets was investigated. The results obtained illustrate the advantages of combining microdialysis with CE-LIFD for studying neurotransmitters with such a high sampling rate.

Detection and enrichment of hematopoietic stem cells by side population phenotype.

A flow cytometric procedure has recently been described to isolate hematopoietic stem cells from mouse bone marrow based on the efflux properties of the vital dye Hoechst 33342. The assay defines a subset of cells-termed the "side population" (SP)-by simultaneously measuring fluorescence of the dye at two wavelengths (~450 nm and >670 nm). In this chapter, SP protocols are provided to detect candidate hematopoietic stem cells in mouse bone marrow and human cord blood. In the standard method, SP profiles are readily observed on a stream-in-air cell sorter using 30 mW of 351-356 nm ultraviolet excitation from a krypton-ion laser. Alternatively, SP profiles can be resolved on an analytical flow cytometer with cuvette flow cell using 8 mW of 325-nm ultraviolet excitation from a helium-cadmium laser. The ability to perform the SP assay on an analytical instrument facilitates optimization of staining conditions to identify hematopoietic and other stem cells in a variety of tissues. It is also demonstrated that SP profiles of slightly lower resolution can be obtained on a stream-in-air cell sorter using 100 mW of 407-nm violet excitation from a krypton-ion laser, raising the possibility that with appropriate validation the SP assay could be performed on flow cytometers that are not equipped with ultraviolet lasers.

Laser-induced autofluorescence microscopy of normal and tumor human colonic tissue

Laser-induced autofluorescence (LIAF) spectroscopy has been found to be a promising tool for early cancer diagnosis in various organs, but the reasons responsible for the spectral differences between normal and diseased tissue are still not well understood. In this study, a microspectrophotometer (MSP) system was used to identify the microscopic origins of tissue autofluorescence in the colon under the excitation of a helium-cadmium laser at 442 nm. Colonic tissue samples (normal: n=8, adenocarcinoma: n=10) were obtained from 12 patients with known or suspected malignancies of the colon. The intrinsic fluorescence spectra and images of fresh tissue sections prepared from normal and tumor colonic tissue were measured by the MSP system. Three distinct tissue layers of the colon were found for fluorescence, the mucosa, the submucosa and the muscularis propria, with submucosa being the most fluorescent. Differences in the spectral shape and intensity of the intrinsic fluorescence originating from different colonic layers indicate that fundamentally different fluorophores may be present in the respective tissue layers. There was no significant difference in the intrinsic fluorescence features of the submucosa between normal and tumor colonic tissue, but the fluorescence intensity of the submucosa in tumor tissue was significantly reduced due to the infiltration of tumor cells into the submucosa. The intrinsic fluorescence spectrum peaking at about 520 nm for tumor stroma appeared more evident than that of normal lamina propria. Limited areas of the lamina propria layer in some adenocarcinoma colon exhibited an emission band at about 635 nm, which was attributed to endogenous porphyrins in tumor. Autofluorescence microscopy revealed that differences in the clinically measured autofluorescence spectra between normal and tumor tissue were mainly due to thickening of the tumor mucosa resulting in a reduced submucosa fluorescence contribution, as well as the increased hemoglobin absorption in tumor tissue. Therefore, investigation of the microscopic origins of tissue autofluorescence and images can provide new insights into morphological structures and biochemical components of tissues, which are vital to improve the implementation of the LIAF technique for non-invasive in vivo tissue diagnostics.

In vivo simultaneous monitoring of gamma-aminobutyric acid, glutamate, and L-aspartate using brain microdialysis and capillary electrophoresis with laser-induced fluorescence detection: Analytical developments and in vitro/in vivo validations.

gamma-Aminobutyric acid (GABA), glutamate (Glu), and L-aspartate (L-Asp) are three major amino acid neurotransmitters in the central nervous system. In this work, a method for the separation of these three neurotransmitters in brain microdialysis samples using a commercially available capillary electrophoresis (CE) system has been developed. Molecules were tagged on their primary amine function with the fluorogene agent naphthalene-2,3-dicarboxaldehyde (NDA), and, after separation by micellar electrokinetic chromatography, were detected by laser-induced fluorescence using a 442 nm helium-cadmium laser. The separation conditions for the analysis of derivatized neurotransmitters in standard solutions and microdialysates have been optimized, and this method has been validated on both pharmacological and analytical basis. The separation of GABA, Glu, and L-Asp takes less than 10 min by using a 75 mmol/L borate buffer, pH 9.2, containing 70 mmol/L SDS and 10 mmol/L hydroxypropyl-beta-cyclodextrin and + 25 kV voltage. The detection limits were 3, 15 nmol/L and, 5 nmol/L for GABA, Glu, and L-Asp, respectively. Moreover, submicroliter samples can be analyzed. This method allows a simple, rapid and accurate measurement of the three amino acid neurotransmitters for the in vivo brain monitoring using microdialysis sampling.

Phonation bronchoscopy

Fluorescence bronchoscopy is one of the methods of the early detection of lung cancer that involves the large airways. The method is based on the detection of the altered autofluorescence of malignantly transformed tissue, and confirmed by biopsy and histopathologic examination. Fluorescence bronchoscopy was performed in 18 patients, mean age of 51.2 years (male n = 12, female n = 6) due to the suspected lung cancer. Fluorescence bronchoscopy was performed using the Xillix LIFE-Lung System, Vancouver, Canada. After conventional white-light bronchoscopy, the tracheobronchial tree was illuminated by blue light (442 nm) using helium-cadmium laser, and the results of autofluorescence were classified into three classes. Normal mucosa was of green fluorescence (Class I), abnormal mucosa was red or dark brown fluorescence (Class II and II), which was the indication for performing biopsy. Normal endoscopy findings were established in 15 patients by conventional bronchoscopy. In the same group, by fluorescence bronchoscopy, Class I of fluorescence (normal finding) was found in 9 patients, while Class II changes occurred in 6 patients. Histopathologic analysis of bronchial mucosa with Class II changes was performed detecting planocellular carcinoma in situ in one patient. Tumor-like changes were detected in 3 patients by conventional bronchoscopy, and were determined as Class III changes by fluorescence bronchoscopy. By the biopsy of these chages carcinoma was documented in 2 patients while in one patient metaplasia of epithelium and granulation tissue around aspirated foreign body was detected. Fluorescence bronchoscopy is one of the methods for detecting metaplasia, carcinoma in situ and cancerous changes of bronchial epithelium in the large airways. However, the high rate of falsely positive findings represents a limitation of this method.

Utilization of a Synthetic Absorbable Sealant Activated by Helium Cadmium Laser Bronchoscopy for Successful Closure of a Persistent Bronchopleural Fistula Following Lobectom

INTRODUCTION: A bronchopleural fistula (BPF) may be one of the most disappointing complications of pneumonectomy or lobectomy. The clinical sequela may include incomplete lung expansion, empyema, prolonged ventilator support, and often gas exchange problems in addition to increased morbidity and mortality. Numerous modalities using chemical agents and mechanical devices have been reported for fistula closure.

Holographic recording mechanisms of gratings in indium oxide films using 325 nm helium-cadmium laser irradiation

UV (325 nm) holographic recording of gratings in indium oxide films fabricated by reactive pulsed laser deposition has been investigated as a function of growth temperature, oxygen pressure and angle of incidence of the plasma plume on the substrate. The influence of the ambient environment (air or vacuum) and the film temperature during recording has also been studied. Large steady state refractive index changes up to 6×10-3 were observed in layers grown at an oblique angle of 75°. About 77% of the magnitude of these changes residues after thermal annealing and is attributed to UV-induced permanent structural rearrangements. In contrast, refractive index changes in films grown at normal incidence were smaller in magnitude and completely reversible.

Photoelectrochemical behavior of poly(3,4-ethylenedioxythiophene) in acetonitrile solutions during cathodic reduction of oxygen

The photoelectrochemical behavior of a polymer electrode based on poly(3,4-ethylenedioxythiophene) (PEDT) is studied in acetonitrile solutions during the cathodic reduction of oxygen. The measurements are taken on PEDT films 490 nm thick deposited onto a platinum support in oxygen-saturated 0.1 M Bu4NBF4 solutions. The electrode was illuminated with a light of a wavelength 487 nm from a helium–cadmium laser in conditions of modulated illumination, and in doing so, the real and imaginary components of an alternating photocurrent were measured at different modulation frequencies. The peculiarity of the photoelectrochemical behavior of PEDT manifests itself in the emergence of an anodic photocurrent, which increases with a change in potential in the cathodic direction. At low modulation frequencies, the real and imaginary components are of the same sign, whereas at high modulation frequencies the components have opposite signs (photocurrent lags behind a change in the light intensity). The emergence of an anodic photocurrent is connected with the cathodic reduction of oxygen and the formation in a near-electrode layer of radical anion O–2. Such a behavior of PEDT, which makes it different from other polymers of the thiophene series, is explained by different potentials of their anodic oxidation, to which a conductive state of the polymer is related.

Photoluminescence studies and read/write process of a strong two-photon absorbing chromophore

The optical properties of a fluorene-based chromophore exhibiting a high two-photon absorption cross section have been investigated both in the pure state and as a guest–host system in poly(N-vinylcarbazole). Upon irradiation with a helium cadmium (He–Cd) laser at 325 nm, the guest–host sample exhibits a blueshift with a maximum emission at 459 nm. Information written onto the samples in the blue can also be read using an Ar+ laser. The chromophore undergoes a chemical change upon irradiation in air, and has been found to no longer exhibit upconversion after this transformation. Infrared analysis of the sample (the chromophore) before and after irradiation was carried out, and the spectra suggest the formation of a new conjugated species. Other applications we have developed using this phenomenon include the successful writing of optical device structures in polymer films where the AF-50 is a guest material.

Neuromuscular plasticity in the locust after permanent removal of an excitatory motoneuron of the extensor tibiae muscle.

The capacity of the larval insect nervous system to compensate for the permanent loss of one of the two excitatory motoneurons innervating a leg muscle was investigated in the locust (Locusta migratoria). In the fourth instar, the fast extensor tibiae (FETi) motoneuron in the mesothoracic ganglion was permanently removed by photoinactivation with a helium-cadmium laser. Subsequently, the animals were allowed to develop into adulthood. When experimental animals were tested as adults after final ecdysis, fast-contracting fibers in the most proximal region of the corresponding extensor muscle, which are normally predominantly innervated by FETi only, uniformly responded to activity of the slow extensor tibiae (SETi) neuron. In adult operated animals, single pulses to SETi elicited large junctional responses in the fibers which resulted in twitch contractions of these fibers similar to the responses to FETi activity in control animals. The total number of muscle fibers, their properties as histochemically determined contractional types (fast and slow), and their distribution were not affected by photoinactivation of FETi. Possible mechanisms enabling the larval neuromuscular system to compensate for the loss of FETi through functionally similar innervation by a different motoneuron, i.e. SETi, are discussed.

3-Aminobenzamide and 3-aminobenzoic acid, tags for capillary electrophoresis of complex carbohydrates with laser-induced fluorescent detection

The efficiencies in derivatization of reducing carbohydrates were compared by capillary electrophoresis using maltose as a model with nine monoaminobenzene derivatives by reductive amination in the presence of sodium cyanoborohydride. We found that aminobenzene derivatives substituted at the 3-position showed good reactivity with reducing carbohydrates as expected from the reaction mechanism, although the fluorescence intensities and molar absorptivities of these derivatives were not as high as those of 2- and 4-aminobenzene derivatives. The reagents, 3-aminobenzamide and 3-aminobenzoic acid, which showed the highest reactivity, were applied to the labeling of carbohydrate chains obtained from some sialic acid-containing glycoprotein samples, and also high-mannose and hybrid-type oligosaccharides. Capillary electrophoresis of these labeled carbohydrate chains in an inner surface-modified capillary with (50% phenyl)methylpolysiloxane allowed excellent separation of sialic acid-containing carbohydrate chains derived from fetuin and thyroglobulin as well as high mannose-type and hybrid-type carbohydrates derived from bovine pancreas ribonuclease B, soybean agglutinin and hen ovalbumin. The lower limit of calibration was as low as the 10 −16 mol (injected amount) with helium–cadmium laser induced detection.