Miura Yoko, Pruessner Joachim, Mertineit Carla Lotta, Kern Katharina, Muenter Michael, Moltmann Moritz, Danicke Veit, Brinkmann Ralf
Institute of Biomedical Optics, University of Luebeck; Medical Laser Center Luebeck GmbH, University of Leubeck; Department of Ophthalmology, University of Luebeck;
Institute of Biomedical Optics, University of Luebeck.
J Vis Exp. 2017 Jun 30(124):54326. doi: 10.3791/54326.
An original method to heat cultured cells using a 1.94 µm continuous-wave thulium laser for biological assessment is introduced here. Thulium laser radiation is strongly absorbed by water, and the cells at the bottom of the culture dish are heated through thermal diffusion. A laser fiber with a diameter of 365 µm is set about 12 cm above the culture dish, without any optics, such that the laser beam diameter is almost equivalent to the inner diameter of the culture dish (30 mm). By keeping a consistent amount of culture medium in each experiment, it is possible to irradiate the cells with a highly reproducible temperature increase. To calibrate the temperature increase and its distribution in one cell culture dish for each power setting, the temperature was measured during 10 s of irradiation at different positions and at the cellular level. The temperature distribution was represented using a mathematical graphics software program, and its pattern across the culture dish was in Gaussian form. After laser irradiation, different biological experiments could be performed to assess temperature-dependent cell responses. In this manuscript, viability staining (i.e., distinguishing live, apoptotic, and dead cells) is introduced to help determine the threshold temperatures for cell apoptosis and death after different points in time. The advantages of this method are the preciseness of the temperature and the time of heating, as well as its high efficiency in heating cells in a whole cell culture dish. Furthermore, it allows for study with a wide variety of temperatures and time durations, which can be well-controlled by a computerized operating system.
本文介绍了一种使用1.94μm连续波铥激光加热培养细胞以进行生物学评估的原创方法。铥激光辐射被水强烈吸收，培养皿底部的细胞通过热扩散被加热。将一根直径为365μm的激光光纤置于培养皿上方约12cm处，不使用任何光学器件，以使激光束直径几乎等同于培养皿的内径（30mm）。通过在每个实验中保持培养基量一致，可以以高度可重复的温度升高照射细胞。为了针对每个功率设置校准一个细胞培养皿中的温度升高及其分布，在不同位置和细胞水平的10s照射期间测量温度。使用数学图形软件程序表示温度分布，其在培养皿上的模式呈高斯形式。激光照射后，可以进行不同的生物学实验以评估温度依赖性细胞反应。在本手稿中，引入了活力染色（即区分活细胞、凋亡细胞和死细胞）以帮助确定不同时间点后细胞凋亡和死亡的阈值温度。该方法的优点是温度和加热时间的精确性，以及在整个细胞培养皿中加热细胞的高效率。此外，它允许在各种温度和持续时间下进行研究，这些可以通过计算机操作系统很好地控制。
