Department of Physics, University of Gothenburg, SE-41296, Gothenburg, Sweden.
Lab Chip. 2010 Mar 7;10(5):617-25. doi: 10.1039/b913587a. Epub 2009 Dec 9.
Cells naturally exist in a dynamic chemical environment, and therefore it is necessary to study cell behaviour under dynamic stimulation conditions in order to understand the signalling transduction pathways regulating the cellular response. However, until recently, experiments looking at the cellular response to chemical stimuli have mainly been performed by adding a stress substance to a population of cells and thus only varying the magnitude of the stress. In this paper we demonstrate an experimental method enabling acquisition of data on the behaviour of single cells upon reversible environmental perturbations, where microfluidics is combined with optical tweezers and fluorescence microscopy. The cells are individually selected and positioned in the measurement region on the bottom surface of the microfluidic device using optical tweezers. The optical tweezers thus enable precise control of the cell density as well as the total number of cells within the measurement region. Consequently, the number of cells in each experiment can be optimized while clusters of cells, that render subsequent image analysis more difficult, can be avoided. The microfluidic device is modelled and demonstrated to enable reliable changes between two different media in less than 2 s. The experimental method is tested by following the cycling of GFP-tagged proteins (Mig1 and Msn2, respectively) between the cytosol and the nucleus in Saccharomyces cerevisiae upon changes in glucose availability.
细胞自然存在于动态的化学环境中，因此有必要在动态刺激条件下研究细胞行为，以了解调节细胞反应的信号转导途径。然而，直到最近，研究细胞对化学刺激的反应的实验主要是通过向细胞群体中添加应激物质来进行的，因此只能改变应激的幅度。本文展示了一种实验方法，该方法能够在可逆环境扰动下获取单个细胞行为的数据，其中微流控技术与光镊和荧光显微镜相结合。使用光镊可以在微流控装置的底部表面上逐个选择和定位细胞，从而实现对细胞密度以及测量区域内细胞总数的精确控制。因此，可以优化每个实验中的细胞数量，同时避免细胞簇的形成，因为细胞簇会使后续的图像分析变得更加困难。对微流控装置进行建模和演示，结果表明其能够在不到 2 秒的时间内可靠地在两种不同的介质之间进行切换。通过在葡萄糖供应变化时观察 GFP 标记蛋白（Mig1 和 Msn2）在酵母细胞的细胞质和细胞核之间的循环，对该实验方法进行了测试。
