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

Lead Research Organisation: University of Cambridge
Department Name: Engineering


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.

Technical Summary

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.

Planned Impact

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.
Description The aim of this grant was to develop a microfluidic device to combine trapping, super-resolution imaging and chromosome structure determination (using a recently developed chromosome conformation capture/PCR amplification technique called single cell Hi-C) of a single mouse embryonic stem cell (mESC). Such a device would allow us to understand how cell cycle and cell differentiation affect how a chromosome folds. In this grant:
1) We have investigated a range of new trapping configurations to efficiently trap and image mESCs (building on previous work in the lab trapping yeast cells).
2) We have modified our existing super-resolution microscopes to carry out 3D super-resolution imaging of single proteins within both live and fixed embryonic stem cells. By generating embryonic stem cell lines expressing fluorophore labeleled proteins, we have shown that this can be used to study the localisation and the dynamic properties of DNA binding proteins. We have also developed software to analyse the localisation of these proteins (applicable to many similar biological problems).
3) We have used our cell trap devices to carry out the initial steps of the chromosome capture technique and have also demonstrated on a separate device that PCR/genome amplification is possible.
Having demonstrated the individual steps of our original plan, the final step awaiting is making the final device that we have designed to incorporate all these steps. During this process, we have trained a graduate student and a postdoc to build these devices, opening up the possibility of developing similar devices for other ESC based studies.
Exploitation Route Academically, We envisage that the results of this proposed project will make possible a systematic approach for tackling similar biological problems. In the short-term, we intend to now build these devices we have designed to understand stem cell chromatin structure and how it changes during the cell cycle and during differentiation. In addition, we hope to take advantage of our ability to to trap and image single stem cells to study single stem cell differences and how they contribute to other processes.

In addition, as our programme is particularly multi-disciplinary in its nature, it involves communication between diverse fields of research, in particular physical chemists, engineers, cell/molecular biologists and computational biologists. The development of this type of research is therefore likely to promote further communication and collaboration between biologists, chemists and mathematical/computational groups.

In the long-term, understanding the differentiation of stem cells towards a committed lineage could have enormous potential for providing a source of human tissue to study disease progression, or to develop drugs for personalised molecular therapies.
Sectors Creative Economy,Digital/Communication/Information Technologies (including Software),Education,Healthcare,Pharmaceuticals and Medical Biotechnology

Description This grant has led to the development of microfluidic devices that can be used for trapping ES cells for super-resolution imaging studies and in a format, enabling subsequent Hi-C analysis. Since most of the work on the project related to technology and tools development, we envisage that most of the impact will come in years to come. We believe that our close collaboration between the biological sciences, the physical sciences, engineering and information technology will help drive technological innovation in this area beyond our proposed grant, leading to both economic and societal impact. So far, it has resulted in the training of a new generation of researchers (one postdoctoral research associate and in linking to one PhD student project), and facilitated in the development of a research capability at the university that can exploit the new technological developments in optical microscopy, microfluidics and modelling of chromatin structure through increased collaboration between the Engineering, Biochemistry and Chemistry Departments. It has led to cell trapping devices, to image analysis software and to the development of a 3D super-resolution microscope that can now be used by the university and other universities. We are also in the process of disseminating our results as widely as possible through university seminars and international workshops and conferences as well as journal publications. Much of the work is being followed up and we foresee it will have an impact on technological development in industry, not just in academia, and hence economic and societal impact over the next few years.
First Year Of Impact 2013
Sector Digital/Communication/Information Technologies (including Software),Education
Impact Types Societal

Title Microfluidic cell traps for super-resolution imaging 
Description An approach to reliably trap ES cells in pre-defined locations to enable super-resolution imaging studies under flow conditions. 
Type Of Material Technology assay or reagent 
Year Produced 2015 
Provided To Others? Yes  
Impact This enables the super-resolution imaging for live ES cells in regulated micro-environments while simultaneously providing the possibility of combining Hi-C protocols on the same microfluidic substrate. 
Description Bioengineering seminar 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Other academic audiences (collaborators, peers etc.)
Results and Impact talk resulted in visibility for our research and follow-on discussions.

the talk resulted in engagement with other local collaborative opportunities.
Year(s) Of Engagement Activity 2014