Microfluidic System for In-Flow Reversible Photoswitching of Near-Infrared Fluorescent Proteins.
Microfluidic System for In-Flow Reversible Photoswitching of Near-Infrared Fluorescent Proteins.
复制标题
用于近红外荧光蛋白的流内可逆光开关的微流体系统。
DOI：
10.1021/acs.analchem.6b03499
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发表时间：
2016
影响因子：
7.4
通讯作者：
Verkhusha,VladislavV
中科院分区：
文献类型：
--
作者：
Lychagov,VladislavV;Shemetov,AntonA;Jimenez,Ralph;Verkhusha,VladislavV
我们已经开发了一个微流控流式细胞术系统，以筛选可逆的光开关荧光蛋白的对比度和稳定性之间的可逆的高和低荧光状态的光转换。用衍射光学器件产生的20个激发和去激活光束的双色阵列与蛇形微流体通道几何形状相结合，该蛇形微流体通道几何形状被设计成提供具有光转换荧光对比度的实时计算的五个光开关循环。利用在近红外（NIR）光谱范围内具有弱荧光的耐辐射球菌光敏色素（DrBphP）液滴，研究了光开关在流中作为激发和失活光束能量密度、流速和蛋白质浓度的函数的特性。与对静止液滴和表达DrBphP的HeLa S3哺乳动物细胞的测量一致，流动系统的优化操作在几赫兹的液滴速率下提供高达50%的光转换对比度，并且在10 000个事件中的变异系数（CV）高达2%。本文建立的微流体流动中亮度和光开关测量的校准方法为筛选基于细胞的可逆开关近红外荧光蛋白文库提供了基础。
We have developed a microfluidic flow cytometry system to screen reversibly photoswitchable fluorescent proteins for contrast and stability of reversible photoconversion between high- and low-fluorescent states. A two-color array of 20 excitation and deactivation beams generated with diffractive optics was combined with a serpentine microfluidic channel geometry designed to provide five cycles of photoswitching with real-time calculation of photoconversion fluorescence contrast. The characteristics of photoswitching in-flow as a function of excitation and deactivation beam fluence, flow speed, and protein concentration were studied with droplets of the bacterial phytochrome fromDeinococcus radiodurans(DrBphP), which is weakly fluorescent in the near-infrared (NIR) spectral range. In agreement with measurements on stationary droplets and HeLa S3 mammalian cells expressing DrBphP, optimized operation of the flow system provided up to 50% photoconversion contrast in-flow at a droplet rate of few hertz and a coefficient of variation (CV) of up to 2% over 10 000 events. The methods for calibrating the brightness and photoswitching measurements in microfluidic flow established here provide a basis for screening of cell-based libraries of reversibly switchable NIR fluorescent proteins.