Microfluidic devices for 3D super-resolution imaging of single molecules in live cells

活细胞中的单分子实验由于各种原因而令人感兴趣：为了能够追踪生命系统中的大分子并量化它们在基因表达调控中的作用，提供对生命系统中基因表达和调节机制的实验见解，并能够提供对生命系统中分子尺度上可能错过的其他随机机制的见解。总体测量通常代表传统工具和接口。该提案建立在初步概念验证实验的基础上，提出了一种结合微流体、纳米级结构和光学检测的方法，为将单细胞分子成像与此类系统中存在的小体积复杂混合物的化学分析相结合开辟了一系列选择。这种独特的技术融合可以为生命系统中独特的单分子研究提供新的平台。这样的系统可以结合联合收割机随机测量的蛋白质分子，使用超分辨率光学显微镜与高通量分析的核结构，以获得真正新颖的洞察到3D组织染色质在活的哺乳动物细胞。所提出的工具将通过在微流体设备内集成适当的捕获结构和光学光片来解决当前存在于将超分辨率成像技术扩展到哺乳动物系统中的限制，以防止导致在这些系统中发现的更大的核结构存在的自发荧光。

Single molecule experiments in living cells are of interest for a variety of reasons: to be able to track macromolecules within living systems and quantify their role in the regulation of gene expression, to provide experimental insight into the mechanism of gene expression and regulation in living systems and to be able to provide insight into other stochastic mechanisms present at a molecular scale in living systems that may be missed in an ensemble measurement typically representative of conventional tools and interfaces. This proposal builds on initial proof-of-concept experiments and presents an approach combining microfluidics, nanoscale structures and optical detection to open up a range of options for integrating single-cell molecular imaging together with chemical analysis of small volumes of complex mixtures present in such systems. This unique convergence of technologies could enable new platforms for unique single-molecule studies in living systems. Such systems could combine stochastic measurements of protein molecules using super-resolution optical microscopy with the high-throughput analysis of nuclear structure to obtain truly novel insight into the 3D organisation of chromatin in live mammalian cells. The proposed tools will address the current limitations that exist in extending super-resolution imaging technology to mammalian systems by integrating appropriate trapping structures and optical light sheets within microfluidic devices to prevent auto-fluorescence that results in the presence of much larger nuclear structures found in these systems.