At present, long term in vitro imaging is costly, limited and closed. With this proposal, we seek funding to support the continued development of a flexible, low cost, liveimaging platform which was initially built to fully quantify the evolution of the cellular network structure and the establishment of the beating dynamics of human cardiomyocytes over extended periods of time, with the ultimate goal to better understand Hypertrophic Cardiomyopathy (HCM).
Background: HCM is the most common form of Mendelianinherited heart disease, affecting approximately 0.2% of the global population and it is the most common cause of sudden cardiac death in individuals younger than 35 years of age. The advent of somatic cell reprogramming to generate human induced pluripotent stem cells (hiPSC) has provided a new model system for studying cellular function and signalling in tissues which would have otherwise required highly invasive procedures from patients. R. Shakur and J. Kadiwala (Sanger Institute) have developed an efficient cardiomyocyte differentiation protocol for hiPSC and are now able to differentiate these cells into spontaneously beating ventricular myocytes that spatially selforganise into an intricate network. Despite this, the role of mechanical transduction and cell migration in the generation of mature electrically stable cardiomyocytes during the differentiation of such cells into beating networks remains unknown, due in most part to the lack of a flexible and affordable commercial imaging solution.
Objectives: The current imaging device comprises an array of lenses/CCD sensors able to monitor up to six wells in a standard plate, is compact (250x200x150mm) enough to fit in an incubator, and requires no additional manual intervention across the monitoring period after setup. With respect to our primary objective, it is anticipated that the present imaging platform will allow us to record the evolution of morphological and dynamic characteristics and analyse them in the light of environmental conditions and cellular states in this model tissue. The work will help optimise the generation of such tissue culture work at high throughput, with direct clinical application for the development of future therapeutics. However, the platform itself should be of wide interest for any application that requires the long term monitoring of cell behaviour in vitro, a technique previously unavailable to the vast majority of synthetic biology labs. The project will in particular address current limitations of this setup and make it much more versatile by including different types of imaging and provide a flexible user interface.
Who we are:
Fergus Riche email@example.com 4th Year engineer in CUED, future Sensors CDT student and participant in 2014 Cambridge/JIC iGEM team. I have been a member of the Cambridge Makespace since 2013, and my interests include rapid prototyping, the application of engineering science to biology and developing measurement instrumentation.
Junead Kadiwala (firstname.lastname@example.org) – Senior research associate in Stem cell biology at University of Cambridge Laboratory of Regenerative Medicine. Has trained many individuals in protocols of derivation and maintenance of human pluripotent stem cells.
Rameen Shakur (email@example.com) – Clinical cardiologist in London and Wellcome Trust clinical fellow at the University of Cambridge based at the Sanger centre and Laboratory of Regenerative Medicine. Specialises in cardiovascular modelling using human IPS cells and model organisms. Application of genome editing and synthetic biology in translational applications of human stem cells.
Alexandre Kabla – firstname.lastname@example.org Senior Lecturer in Engineering. Research interests include mechanobiology of cell populations and developmental biology, instrumentation and computer modelling.
Ongoing work has so far produced a basic proof of concept device which consists of six identical transilluminated brightfield microscopes, where illumination is provided by broad spectrum white LEDs.
The illumination intensity can be computer controlled, but the optical flexibility of the illuminator is limited, consisting of a fixed aperture experimentally optimised to maximise contrast whilst maintaining resolution when imaging human fibroblast cells. The microscopes are mounted in an array on a frame with manual focus adjustment screws, which accepts standard size cell culture plates and can be placed in an incubator for long term imaging applications. Images and short videos are collected using Raspberry Pi (RPi) camera modules and boards, which are mounted in a separate enclosure and locally networked in order to coordinate the image capture process. With the SynBio grant, would use this device as a starting point to develop a more flexible generic optical monitoring system for biological applications. Methods: Optics Whilst the brightfield setup produces acceptable results, fluorescenceimaging is a far more widely used vital imaging technique that produces superior contrast and resolution, which can be used to track sub cellular structures and processes by the use of localisation tags and inducible promoters. An initial transmission fluorescence imaging setup as been developed with promising results on fluorescein coated paper, utilising an LED illumination source, single condenser lens and manually variable aperture. It would be our intention to extend this setup to the entire array, introduce interchangeable filters for different fluorophores, and automate the aperture control. In addition to fluorescence microscopy, we intend to develop a phase contrast setup which would require the development of a more sophisticated illumination array, and would allow the imaging of wild type and nonfluorescent cells. User interface To complement the automation of the image capture, we would develop software and hardware tools to automate the focus and position of the microscope array, as well as the aperture size and illumination intensity, such that these variables can be set more precisely and flexibly. In addition, we will integrate with the openlabtools web interface (https://github.com/OpenLabTools/OpenLabToolswebinterface) in order to allow data to be collected remotely, and changes to the setup to be made dynamically during the imaging cycle. Data analysis In addition to the work that is to be carried out over the next two months on analysing the results from stem > cardiomyocyte differentiation, further work will be undertaken in order to develop and calculate position and movement metrics from visual timelapse data of cells. This part of the project will be more specific to the particular research study the original microscope array was developed for, but is critical to demonstrate that the tool can be used to produce research quality data. In addition, the data analysis functions will be documented and made available online to allow collaboration and modification for related morphology applications.
Benefits and outcomes:
Tangible outcome which can readily be shared : A kit fluorescence/ brightfield/phase contrast microscope array which can be easily assembled and operated by a remote user. This project aims to construct a low cost open source modular microscope array, suitable for imaging specimens in an incubator for extended (48hrs+) periods of time. By providing full developmental documentation , users will be able to make well justified modifications to the platform to suit their individual needs. Promote interdisciplinary working and exchange
The project is carried by a multidisciplinary team, and has elements of mechanical, optical and computational design in order to solve a problem in the life sciences. By incorporating end user feedback in parallel with the development, we will be able to make the platform both feature rich and easy to use.
Relevant to synthetic biology and provide a valuable contribution to the field
This device and the associated software will automate the routine aspects of the quantitative study of morphogenesis and cellular processes over a range of timescales, which would otherwise be prohibitively costly on a time and resource basis. The platform seeks to invert the current commercial paradigm of adding an environmentally controlled area to a microscope by adding imaging facilities to an incubator, where space is at far less of a premium and more measurements can be taken.
Address focus areas of the SRI e.g. open technologies In addition to the relatively low cost of the device, its assembly will require no more than a laser cutter, soldering iron and saw, all of which are widely available tools in universities or local community-operated hackerspaces. Design files and quality instructions for assembly, operation and modification will be released online.
The basis of the project has already been developed, and is currently in testing on stem>cardiomyocyte cell differentiation. Adding fluorescence and remote management is realistic considering the experience and preliminary results already acquired by the team. We are not sure at this stage how quantitative the measurements can be, mostly due to the limitations of the RPi camera sensor firmware, but it is anticipated that future versions of the firmware will allow extraction of the raw data from the camera sensor, enabling quantitative illumination intensity measurements to be made.
External Collaboration and matched funding:
The current project is a collaboration between CUED and the Sanger Institute. Matching funding will be provided by the Engineering Department through an Engineering for the Clinical Practice grant (£10k), started in the summer 2014. These funds have already provided support for the development of the brightfield system currently under test. If we are awarded a grant from the SynBio Fund, CUED will financially support Fergus Riche over the summer to develop the fluorescence setup and its web based access, and cover the hardware/consumables costs exceeding the £4k allocated by the SynBio grant.
Optics, fluorescence filters and light sources ~ £3000 Hardware, incl Electronics Materials ~ £500
Biological Materials ~ £1000 3D printing (Digits 2 Widgets, 3dprintuk, CU Architecture Dept.) ~ £300
Makespace Access ~ £100 See also "External Collaboration and matched funding" section of "Benefits and outcomes"