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

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.

This proposal outlines an approach towards developing novel microfluidic devices that will allow 3D super-resolution photo-activated fluorescence microscopy (PALM/STORM) of single molecules in live mammalian cells. The microfabricated devices will be integrated with environmental controls, fluidic handling and optical elements to enable new modalities for such studies. Furthermore, 3D fluorescence imaging will be combined with chip-scale chromosome conformation capture analysis by integrating the appropriate chemical analysis steps together with whole genome DNA amplification (WGA) on the same microfluidic substrate together with an efficient sample extraction system for high-throughput DNA sequencing. 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 single mammalian cells.

This proposal leverages a long-standing ongoing collaboration between the Laue and Seshia groups at Cambridge University to address the development of a microfluidic platform that enables the integration of stochastic measurements of protein molecules using super-resolution optical microscopy together with the high-throughput analysis of nuclear structure to obtain truly novel insight into the 3D organisation of chromatin in single mammalian cells. The proposal builds on very early-stage collaborative proof-of-concept experiments that have shown the validity of this approach in the context of single molecule live cell imaging. These tools are of interest in the context of single molecule experiments to study gene expression in live single cells under controlled microenvironmental cues, studying the link between the 3D structural conformation of chromatin on gene expression and providing a basis to help address fundamental questions underlying the epigenetics of disease processes. In the longer term, the devices and methodologies developed in this work may also be extended to provide a platform for studying the differentiation of embryonic stem cells under controlled microenvironmental cues.

To ensure that the methods developed in this project are of any use - we will focus on ensuring that the tools and methods developed in this project can be readily transferred to other research groups working in similar areas through a number of dissemination and networking activities that are expected to continue throughout the project. These will link to established University and international academic and research networks in the diverse range of disciplines constituting the project (structural biology, genomics, microfluidics and lab-on-chip devices, single molecule experiments, live cell imaging). Both investigators have an established track record of publishing in high-impact scientific journals and international conferences as well as other training and dissemination activities that will be pursued in the context of this project as well. Deliverables such as CAD files and technical user reports will be disseminated through online repositories so that the devices developed under the project may be used by other research groups worldwide.

While immediate translational opportunities are not expected to arise within the project lifetime, efforts will be made to ensure that all follow-on opportunities to spin-off research outcomes are actively pursued. It should be noted that both investigators have a number of links with companies spanning the space from translational genomics and personalised medicine (e.g. Horizon Discovery Ltd.), gene chips and single molecule platforms (e.g. Base 4 innovation), optical microscopy (e.g. Olympus Microscopy) and microfluidics and lab-on-chip devices (e.g. Micronit Microfluidics BV and IME A*STAR Singapore) and opportunities to develop these links further in the context of translational and follow-on collaborative activities to translate the tools and technologies developed under this project will be pursued as appropriate.