Upgrading a microplate reader for photobiology and all-optical experiments
Upgrading a microplate reader for photobiology and all-optical experiments
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DOI：
10.1039/c4pp00361f
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发表时间：
2015-01-01
影响因子：
3.1
通讯作者：
Moeglich, Andreas
中科院分区：
文献类型：
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作者：
Richter, Florian;Scheib, Ulrike S.;Moeglich, Andreas
自动化可以极大地降低实验劳动力成本，从而促进高实验通量，但用于照明实验自动化的现成硬件在商业上却很少。在这里，我们使用廉价的开源电子设备为多模式酶标仪添加可编程照明功能。我们部署此设置来表征三种不同感觉感光器中的光触发现象。首先，我们研究了不同波长的光对拟南芥光敏色素 B 的光活化作用。其次，我们研究了集胞藻的暗态恢复动力学。多种温度和咪唑浓度下的蓝光传感器 Slr1694；虽然咪唑强烈加速了Slr1694 W91F突变体的动力学，但野生型蛋白几乎不受影响。第三，我们确定了 Beggiatoa sp 的光响应。中国仓鼠卵巢细胞中的光激活腺苷酸环化酶 bPAC。 bPAC 以剂量依赖性方式被蓝光激活，半最大强度为 0.58 mW cm (2)； bPAC 激活时产生的细胞内 cAMP 尖峰在光关闭后衰减，半衰期约为 5 分钟。总而言之，我们提出了一种易于组装的装置，从而提供了一种以高通量、可重复性和保真度进行照明实验的简便方法。
Automation can vastly reduce the cost of experimental labor and thus facilitate high experimental throughput, but little off-the-shelf hardware for the automation of illumination experiments is commercially available. Here, we use inexpensive open-source electronics to add programmable illumination capabilities to a multimode microplate reader. We deploy this setup to characterize light-triggered phenomena in three different sensory photoreceptors. First, we study the photoactivation of Arabidopsis thaliana phytochrome B by light of different wavelengths. Second, we investigate the dark-state recovery kinetics of the Synechocystis sp. blue-light sensor Slr1694 at multiple temperatures and imidazole concentrations; while the kinetics of the W91F mutant of Slr1694 are strongly accelerated by imidazole, the wild-type protein is hardly affected. Third, we determine the light response of the Beggiatoa sp. photoactivatable adenylate cyclase bPAC in Chinese hamster ovary cells. bPAC is activated by blue light in dose-dependent manner with a half-maximal intensity of 0.58 mW cm (2); intracellular cAMP spikes generated upon bPAC activation decay with a half time of about 5 minutes after light switch-off. Taken together, we present a setup which is easily assembled and which thus offers a facile approach to conducting illumination experiments at high throughput, reproducibility and fidelity.