Schneckenburger Herbert, Hendinger Anita, Sailer Reinhard, Strauss Wolfgang S L, Schmitt Michael
Institut für Angewandte Forschung, Fachhochschule Aalen, 73428 Aalen, Germany.
J Biomed Opt. 2002 Jul;7(3):410-6. doi: 10.1117/1.1485758.
The plasma membrane of Chinese hamster ovary cells was made permeable using the focused beam of an argon ion laser (488 nm) and phenol red as a light absorbing dye. Small circular dark spots on the cell surface appeared immediately after laser irradiation and disappeared within about 5 min. They were related to transient changes in membrane properties, which could be visualized using the fluorescent marker laurdan, and were probably due to a local increase in temperature. According to a colony forming assay, cell viability was maintained by using light doses up to 2.5 MJ/cm(2) applied for 1 s. In addition to measurements of the efflux of the cytoplasmic marker calcein, cell transfection using a green fluorescent protein (GFP) coding plasmid was studied: brightly fluorescent GFP with an emission maximum around 510 nm was observed within part of the cells after 24 h. The transfection rates after laser irradiation were around 30% for younger subcultures and less than 10% for aging cells. This may be due to age dependent changes in the phase transition of membrane lipids from gel phase to liquid crystalline phase. High transfection rates, visual control and universality towards various cell lines are possibly the main advantages of laser-assisted optoporation in comparison with presently existing methods of cell transfection.
利用氩离子激光（488nm）的聚焦光束和作为光吸收染料的酚红，使中国仓鼠卵巢细胞的质膜具有通透性。激光照射后，细胞表面立即出现小的圆形暗斑，并在约5分钟内消失。它们与膜性质的瞬时变化有关，这种变化可用荧光标记物劳丹明可视化，可能是由于局部温度升高所致。根据集落形成试验，使用1秒内施加高达2.5MJ/cm² 的光剂量可维持细胞活力。除了测量细胞质标记物钙黄绿素的流出外，还研究了使用绿色荧光蛋白（GFP）编码质粒进行细胞转染：24小时后，在部分细胞内观察到发射最大值约为510nm的明亮荧光GFP。激光照射后的转染率对于较年轻的传代培养物约为30%，对于衰老细胞则小于10%。这可能是由于膜脂从凝胶相到液晶相的相变存在年龄依赖性变化。与目前现有的细胞转染方法相比，高转染率、可视化控制以及对各种细胞系的通用性可能是激光辅助光穿孔的主要优点。
