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ePaper created 2017-03-17, 12:41:55 | version 1.38.0
% / y t i l a h t e L 35 30 25 20 15 10 5 0 34.7 DPSC cells CHO cells 14.5 7.2 26 Laser power / mW 0 20 transferred to an incubator in order to guarantee optimal conditions for further growth, cell division and repairing processes. Pulse-stretching unit, generation and measure- ment of 700 fs pulses In order to increase the pulse duration at the sam- ple to 700 fs, a pulse-streching unit was imple- mented in the light path in front of the microscope. This unit consists of coated mirrors and two parallel arranged gold-sputtered gratings with a grating constant of 600 lines/mm. The second grating was mounted on a motorised stage with micrometre pre- cision. The pulse width was varied in dependence on the grating distance. The laser beam received a spa- tial dispersion by the first grating, which was com- pensated at the second grating (Fig. 1). The pulse duration was initially determined at the laser exit with the autocorrelator MINI (APE, Berlin, Germany) with 88 fs at a central emission wave- length of 750 nm, 80 fs at 800 nm and 91 fs at 850 nm, hypothesising a Gaussian function. In gen- eral, measurements at the focal plane of the objec- tive proved difficult, as divergent beams exist. A flat, non-linear measurement diode was employed, thus facilitating the measurement at the focal plane of high-aperture objectives. The autocorrelation func- tion (ACF), which can be fitted with either Gauss-, Lorentz or Secanthyperbolicus-based analysis pro- grammes in order to calculate the pulse duration. Life-/Dead-Test In order to examine the vitality of dental pulp stem cells (DPSC), a test by Molecular Probes (Eugene, Oregon, USA) was applied. A mixture of 2 µM cal- cein AM and 4 µM ethidium-homodimer-D1 was added to the cell chambers and incubated for 20 min at 37 °C. Live cells were stained by calcein (emission in the green spectrum), dead cells by ethidium-homodi- mer-D1 with an emission in the red spectrum (nu- cleus). Calcein AM is a non-fluorescent dye which easily permeates the cell membrane of live cells and is transformed by an enzymatic reaction to the strongly green fluorescent calcein which cannot pass the intact membrane. Ethidium-homodimer-D1 is a red fluorescent so-called dead-cell staining agent which can only permeate damaged cell mem- branes and is significantly intensified by binding to DNA. The Life-/Dead-Kit was incubated 5.5 h after irradiation. Fluorescence was achieved by two-pho- ton excitation. Verification of laser-induced ROS-formation The formation of ROS was verified in situ via two-photon excitation of the membrane-perme- able fluorophore dihydroflurescein (DHF) according to the method by Hockberger et al.2 First, the cells were incubated with the marker (10 µM, Fluka, Ger- many) and irradiated after 15 min incubation time. Only one ROI (region of interest) was subjected to irradiation. Surrounding cells were used as control. After irradiation, a full-frame scan was realised with the low power of 4 mW in order to visualise the effect. Results The pulse-stretching unit was adjusted in a way that allowed for each laser wavelength a pulse du- ration of 700 fs ± 50 fs in the focus. Single DPSC cells were scanned 10 x and the effect compared to the non-irradiated surrounding cells was deter- mined. In addition, comparative experiments were accomplished using CHO cells. Cells or cell clusters were selected which were widely spaced in order to inhibit any intercellular communication as far as possible. During scanning, the transmission signal was detected and displayed as a picture on the monitor. A total of 325 cells was subjected to mor- phological examinations as well as a life-/dead- test, 50 cells underwent ROS examination. The re- sults were compared to earlier findings with a pulse width of 170 fs. Morphological changes So-called black spots resulted from specific irra- diation power perameters in locations with signifi- cant granulation in the cells. The laser power was gradually increased by 2 mW in order to measure the threshold value for the appearance of the first la- ser-induced morphological changes (Tab. 1). Under these conditions and with optimum focus, the min- imal power for the appearance of black spots was 20 mW for the DPSC cells and 22 mW for the CHO cells (Tab. 2). At a power of 26 mW, 30 % of the DPSC cells and only 15 % of the CHO cells presented these morphological changes. Cells with laser-induced black spots generally revealed morphological changes within the following five hours thus indi- cating a photodamage effect. research | Fig. 2: Laser-induced damage rate (lethality). laser 1 2017 19