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ePaper created 2017-11-24, 11:00:53 | version 1.38.0
Individual cells were marked by special diamond engravings in the exterior glass window. In case of the detection of cellular damage, these engravings were easily located by applying the phase-contrast technique. Laser microscopy An 80 MHz Ti:Sa Laser, Mai Tai (Spectra-Physics, Mountain View, USA) was applied for femtosec- ond-laser microscopy in the near infrared (NIR) spectrum. The laser power at microscope entrance and objective plane (power at the sample) were de- termined by the measuring instrument Fieldmas- ter (Coherent, Santa Clara, USA) and the measuring head LM2 and varied by grey filters when neces- sary. The measured values were specified as cor- rected in the presented protocol. This correction results from a limited measurement area and al- tered radiation conditions in the medium when compared to air. Laser microscopy was realised via a modified LSM 410 (ZEISS, Jena, Germany) with a 40 x/1.3 oil immer- sion objective. The microscope scan modus 512 x 512 with a laser scan time t = 16 s was applied for cell ir- radiation. The cells were scanned ten times at the same focus plane. These experiments were realised at a central wavelength of 800 nm. After ir radiation, the cells were transferred to an incubator in order to guarantee optimal conditions for further growth, cell division and repairing processes. Pulse-stretching unit, generation and measurement 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 wavelength of 750 nm, 80 fs at 800 nm and 91 fs at 850 nm, hypothesising a Gaussian function. In general, measurements at the focal plane of the objective proved difficult, as divergent beams ex- ist. A flat, non-linear measurement diode was em- ployed, thus facilitating the measurement at the focal plane of high-aperture objectives. The auto- correlation function (ACF), which can be fitted with either Gauss-, Lorentz- or Secanthyperboli- photodamage of pulpa cells research | 35 30 25 20 15 10 5 0 % / y t i l a h t e L 34.7 DPSC cells CHO cells 14.5 7.2 0 20 26 Laser power / mW cus-based analysis programmes in order to calcu- late the pulse duration. Fig. 2: Laser-induced damage rate (lethality). 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-homodi- mer-D1 is a red fluorescent so-called dead-cell staining agent which can only permeate damaged cell membranes and is significantly intensified by binding to DNA. The Life-/Dead-Kit was incubated 5.5 h after irradiation. Fluorescence was achieved by two-photon excitation. Verification of laser-induced ROS-formation The formation of ROS was verified in situ via two-photon excitation of the membrane-permeable fluorophore dihydroflurescein (DHF) according to the method by Hockberger et al.2 First, the cells were incubated with the marker (10 µM, Fluka, Germany) and irradiated after 15 min incubation time. Only one ROI (region of interest) was subjected to irradiation. Surrounding cells were used as control. After irradi- ation, a full-frame scan was realised with the low power of 4 mW in order to visualise the effect. roots 4 2017 11