A CE-MALDI interface based on the use of prestructured sample supports.

We have developed an off-line coupling of capillary electrophoresis (CE) to matrix-assisted laser desorption/ionization time-of-flight mass spectrometry(MALDI-TOF-MS) based on CE fraction collection onto prestructured MALDI sample supports. Analyte carryover and detection sensitivity were investigated using a standard peptide mixture. Low femtomole amounts were detected, and no noticeable carryover was discovered. The performance of the method was evaluated with a mixture of tryptic digests of proteins from a human fetal brain cDNA expression library. The total number of identified peptides was increased from 47 to 211 when the CE-MALDI interface was used compared to direct MALDI-MS analysis. Sequence coverage with CE-MALDI was in the 25-60% range for the different proteins, corresponding to an increase of 1.3-4.9 times relative to that obtained with MALDI-MS of the crude mixture. Fractionation of sample components also facilitated protein identification by MALDI postsource decay analysis. Our initial results suggest this CE-MALDI interface can be used for the analysis of complex peptide mixtures isolated from biological tissues.

Laser desorption/ionization mass spectrometry of peptides and proteins with particle suspension matrixes.

Systematic investigations of particle suspensions for the laser desorption/ionization of peptides and proteins are presented. The performance and suitability for time-of-flight mass spectrometry of different particle materials and sizes, suspended in a variety of different liquids, are described. Performance characteristics such as accessible mass range, achievable mass resolution, analytical sensitivity, and fragmentation are reported. For the desorption of peptides and small proteins, nanoparticle suspensions in glycerol were found to perform comparably to UV-MALDI-MS with common "chemical" matrixes. For proteins in the mass range of ∼12-30 kDa, mass resolution and analytical sensitivity decrease sizeably; for proteins with masses in excess of ∼30 kDa, no spectra could be recorded with any of the tested particle/liquid combinations. The results were found to be largely independent of the laser wavelength in the range from the near-UV to the near-IR because of the strong particle absorption throughout this wavelength range. Ions are shown to originate predominantly from analyte molecules adsorbed at the particle surface. Nanoparticles with a diameter of a few nanometers were found to be superior to microparticles of ∼1 µm diameter or above. Thermodynamic modeling suggests that this different behavior is caused by the different achieved peak temperatures of the two particle sizes.

Fluence and mutagenic side effects of excimer laser radiation applied in ophthalmology in human lymphocytes in vitro.

PURPOSE: To investigate the influence of different fluences in 193 and 248 nm excimer laser radiation on the yields of chromatid and chromosome aberrations induced in human lymphocytes in vitro. METHOD: Heparinized human blood was exposed to 193 or 248 nm excimer laser radiation. The fluence was gradually increased from 21 to 400 mJ/cm2 in 193 nm (constant total energy of 250 J) and from 150 to 377 mJ/cm2 in 248 nm radiation (constant total energy of 500 J). Chromatid and chromosome aberrations were then analysed microscopically. RESULTS: The yields of chromatid breaks and achromatic lesions depend on the fluence per pulse. This dependence contains a linear component, indicating a threshold of about 70 mJ/cm2 fluence in 193 nm and of about 250 mJ/cm2 fluence in 248 nm laser radiation. An increase of the yield of dicentric chromosomes could only be observed at the highest fluence tested (377 mJ/cm2) in the 248 nm series. Over 126 mJ/cm2 in 193 nm radiation no lymphocytes could be cultured and therefore no aberrations could be found. CONCLUSIONS: Our findings show that the fluence of 193 nm and of 248 nm excimer laser radiation has an effect on the yields of chromatid breaks and achromatic lesions in human lymphocytes under in vitro conditions.

The morphological tissue response of the piglet oesophagus to experimental irradiation by 1320 nm Nd:YAG laser.

The oesophagus of 18 minipiglets was exposed to endoscopic intraluminal irradiation with a 1320 nm Nd:YAG laser (10 W, 20 s) via a radial applicator with strictly radially symmetric light distribution. Immediately and at 2, 3, 4 and 8 wk after irradiation, the oesophagus was perfusion-fixed and filled with contrast solution. Radiographs were taken for evaluation by microradiometry. The specimens were subsequently prepared for light and transmission electron microscopy. The immediate reaction to irradiation was a morphological gradient of damage extending from the centre of the laser exposure where there was cellular thermonecrosis in all layers of the wall and condensation of the extracellular matrix, to a peripheral zone (at a distance of up to 8 mm from the region where the laser was centred) which showed only minor tissue damage manifested by intracellular vacuolation. In this zone dilatation of most vascular lumina was prominent. In the period between 2 and 8 wk after irradiation all phases of wound healing were observed and resulted in occlusion of the lumen of the oesophagus by early scar tissue after an interval of more than 3 wk at the former centre of laser exposure. Peripherally, epithelial regeneration resulted in a new luminal lining. Both the process of epithelial regeneration and that of fibrous repair indicated a good reparative capacity of laser-irradiated oesophageal wall tissues resembling the phases of normal wound healing. The immediate laser interaction with tissue indicated that the noncellular matrix components of tissues are more resistant to the photothermal effect than the cellular components.

The effect of ArF-excimer laser irradiation of the human enamel surface on the bond strength of orthodontic appliances.

This study investigated enamel laser conditioning as an alternative to acid etching in bracket therapy. In preliminary experiments optimal laser parameters for achieving a bond strength of 6-10 N/mm2 were defined. Enamel surface morphology was assessed and the ablation depth was measured on serial enamel sections. Thirty human molars were exposed to 193 nm ArF-excimer laser radiation (energy density: 260 mJ/cm2) by single pulse application of 23 nanoseconds. Thirty molars were etched with phosphoric acid (37%) for 60 seconds. The brackets from the treated molars and 30 untreated molars were debonded vertically for tensile bond strength measurement. Roughened enamel surfaces were attained by 450 and 900 laser pulses with a mean ablation depth of 10.13 +/- 4.84 microns. After 1-10 laser pulses, the enamel surface appeared intact. The tensile bond strength was 6.63 +/- 2.18 N/mm2 in the laser-treated group (1 pulse), 8.75 +/- 3.61 N/mm2 in the acid-etched group, and 4.61 +/- 3.15 N/mm2 in the untreated group. We conclude a laser-selective ablation of the membranous enamel pellicle. Since the irradiated area can be adapted to bracket base and the enamel surface remains morphologically intact, pulsed ArF-excimer laser treatment seems to be superior to the acid etching technique.

[The morphological results of the use of a pulsed krypton fluoride excimer laser for enamel conditioning]. / Morphologische Ergebnisse zur Anwendbarkeit eines gepulsten Kryptonfluorid-Excimerlasers im Rahmen der Schmelzkonditionierung.

Although there have been many advances in the field of acid etching, major problems still present. For the most part, these are the unpredictability of the effects of etching, the lack of control of the depth of etching over the long term, and the development of enamel fractures occurring on debonding. The aim of this study was to investigate the morphological effects of enamel conditioning using a pulsed krypton fluoride excimer laser (wavelength 248 nm). Scanning electron microscopic examination revealed a surface similar to that produced by peripheral etching; no signs of thermal damage were to be seen. The requirements that have to be met by a modern laser considered as a possible alternative to conventional acid etching, are discussed.