Karl Stock

Fluorescence lifetime imaging (FLIM) of rhodamine 123 in living cells

A novel setup for fluorescence intensity and lifetime imaging (FLIM) of living cells is reported. Time-resolving techniques are combined with total internal reflection fluorescence microscopy (TIRFM), which permits optical excitation of either plasma membranes or whole cells depending on whether the angle of incidence of the excitation light is greater or smaller than the critical angle for total internal reflection. The method is applied to BKEz-7 endothelial cells incubated with various concentrations of the well established mitochondrial marker rhodamine 123(R123). Measurements show that only at low concentrations this dye is mainly located within the mitochondria, whereas at higher concentrations an accumulation within the plasma membrane occurs as well. Concomitantly, fluorescence quenching in the mitochondria is observed at high concentrations, probably due to aggregation of the R123 molecules. Therefore, for diagnostic applications the concentration of R123 in the incubation medium should not be above 25 microM.

Comparison of Er:YAG and Er:YSGG laser ablation of dental hard tissues

To compare ablation quality of Er:YAG and Er:YSGG laser the surface quality, crater shape, mass loss, and temperature development were determined using the same fiber transmission system and handpiece. Similar crater depths for both lasers but greater diameters for the Er:YAG laser were measured. Also mass loss per pulse of the Er:YAG laser exceeds that of the Er:YSGG laser. Temperature development while ablation of dentin is more pronounced for the Er:YSGG laser. The observed minor ablation quality of the Er:YSGG laser can be explained by the lower absorption coefficient of dental hard substances compared to the Er:YAG laser.

Er:YAG removal of subgingival calculi: efficiency, temperature, and surface quality

Aim of this study was to evaluate a handpiece with a special fiber tip for Er:YAG laser removal of subgingival calculus. The morphological changes of the treated areas were observed by light microscopy and SEM. To evaluate the efficiency, the time for complete removal of the calculi was measured. The temperature development in the pulp during laser treatment was investigated by a thermocouple inserted into a root canal. After calculi ablation, only smooth ablation traces are visible. The depth of the ablation traces is about 100 micrometers . Roughness of the treated area is similar to the untreated area. The ablation efficiency depends strongly on the shape and location of the calculi and varies between 4.6 and 20 mm2/min. During non stop preparation the maximum occurred temperature in pulp was 7.8 K. The results show that the Er:YAG laser with the used fiber delivery systems and handpiece is suitable for effective and sparing removal of subgingival calculus in periodontal treatment.

Morphology of Er:YAG-laser-treated root surfaces

From previous studies it could be demonstrated that an efficient ablation of dental calculus is possible using an Er:YAG laser with a special contact fiber tip. After improving of the design and the efficiency of light transmission of the contact tip laser treated tooth root surfaces were investigated due to morphological changes in comparison to conventional root scaling and planing. Surface modifications were observed histologically under the light microscope and by means of a Scanning Electron Microscope. During laser treatment the intrapulpal temperature increase was measured. The results show that the improved contact tip a microstructured surface can be generated, which shows no signs of thermal effects even when a laser pulse repetition rate of 15 Hz was used. Temperature increase was limited to 4 K at a repetition rate of 10 Hz and to 5.5 K at a repetition rate of 15 Hz.

Controlled tooth surface heating and sterilization by Er:YAG laser radiation

Previous studies on thermal effects of Er:YAG laser tooth irradiation have considered heat as an unwanted side effect of tissue ablation.In contrast, we now investigate the potential of the Er:YAG laser for controlled heating and surface sterilization. The methods include model calculations, temperature measurements, and the investigation of bactericidal effects on natural carious lesions. Due to the strong absorption, in the initial phase of subablative irradiation, there is steep temperature gradient in the tissue, subsequently fading off by heat conduction. Using repetitive pulses and irradiation times of a few seconds, it is possible to reach a surface temperature increase (Delta) T oscillating between 100 K and a few 100 K, while keeping (Delta) T in 1.5 mm depth below 6 K. Carious surfaces can be completely sterilized. Thus the Er:YAG laser offers a new potential for controlled and sparing sterilization in dentistry.

Er:YAG laser for endodontics: efficiency and safety

Recently it has been shown that bacterias can be sterilized by Er:YAG laser irradiation. By optical fiber transmission the bactericidal effect can also be used in endodontics. In order to explore potential laser parameters, we further investigated sterilization of caries and measured temperatures in models simulating endodontic treatment. It was found out that the bactericidal effect is cumulative, with single pulses being active. This offers to choose all laser parameters except pulse energy (radiant exposure) from technical, practical or safety considerations. For clinical studies the following parameter set is proposed for efficient and safe application (teeth with a root wall thickness > 1 mm, and prepared up to ISO 50): pulse energy: 50 mJ, repetition rate: 15 Hz, fiber withdrawal velocity: 2 mm/s. With these settings 4 passes must be performed to accumulate the total dose for sterilization.

Time-resolved in situ measurement of mitochondrial malfunction by energy transfer spectroscopy

To establish optical in situ detection of mitochondrial malfunction, nonradiative energy transfer from the coenzyme NADH to the mitochondrial marker rhodamine 123 (R123) was examined. Dual excitation of R123 via energy transfer from excited NADH molecules as well as by direct absorption of light results in two fluorescence signals whose ratio is a measure of mitochondrial NADH. A screening system was developed in which these signals are detected simultaneously using a time-gated (nanosecond) technique for energy transfer measurements and a frequency selective technique for direct excitation and fluorescence monitoring of R123. Optical and electronic components of the apparatus are described, and results obtained from cultivated endothelial cells are reported. The ratio of fluorescence intensities excited in the near ultraviolet and blue-green spectral ranges increased by a factor 1.5 or 1.35 after inhibition of the mitochondrial respiratory chain by rotenone at cytotoxic or noncytotoxic concentrations, respectively. Concomitantly the amount of mitochondrial NADH increased. Excellent linearity between the number of cells incubated with R123 and fluorescence intensity was found in suspension.

Q-switching of Er:YAG (2.9 /spl mu/m) solid-state laser by PLZT electrooptic modulator

Small-size PLZT 8.5/65/35 ceramic modulators with the length of 4-6 mm are used to obtain Q-switched pulses of the Er:YAG lasers-the pulsewidth 150 ns, energy of the single pulse up to 5 mJ, if followed by a number of the small-intensity postlasing pulses-the total energy of pulses is up to 10 mJ.

Comparison of different focusing fiber tips for improved oral diode laser surgery.

State of the art for use of the fiber guided diode laser in dental therapy is the application of bare fibers. A novel concept with delivery fiber and exchangeable fiber tips enables the use of tips with special and optimized geometries for various applications. The aim of this study is the comparison of different focusing fiber tips for enhanced cutting efficacy in oral surgery.

Mechanism of high-power NIR laser bacteria inactivation.

Lasers are used in dentistry for a variety of indications. One of these is the disinfection of root canals or the sterilization of residual caries. Many studies have demonstrated the capacity to kill bacteria for lasers but the fundamental mechanism of the laser effect remains quite unclear. With our experiments we wanted to determine whether high-power NIR laser bacterial killing is caused by the light itself (photochemical effect) or by a photothermal process. In order to differentiate between mechanisms we heated bacteria suspensions of a nonpathogenic strain of E. coli by a water bath and by a diode laser (940 nm) with the same temporal temperature course. Furthermore, bacteria suspensions were irradiated while the temperature was fixed by ice water. Killing of bacteria was measured via fluorescence labelling. Comparison of killing rates between laser and water-based heating shows no significant differences. The most important parameter is the maximum temperature. Laser irradiation of bacteria at low temperatures does not result in killing. Our experiments show that at least for E. coli bacteria inactivation by high-power laser irradiation is solely based on a thermal process.