Community Research and Development Information Service - CORDIS



Project ID: 665233
Funded under: H2020-EU.1.2.1.

Periodic Reporting for period 1 - CHROMAVISION (Super-resolution visualisation and manipulation of metaphase chromosomes)

Reporting period: 2015-06-01 to 2016-05-31

Summary of the context and overall objectives of the project

CHROMAVISION aims to develop a pioneering chromosome imaging and manipulation platform that will fuel the next decades of structural chromosome research. Chromosomal abnormalities are characteristic of many disorders such as cancer, impaired fertility due to maternal aging, and neurological disorders such as fragile X syndrome. If humanity is to fully understand the wide range of diseases that are associated to errors in cell division, we must be able to further 'zoom in' on healthy and diseased chromosomes in all their complexity. The CHROMAVISION platform will allow molecular biologists to automatically isolate individual chromosomes from small tissue or cell samples and have these delivered to a super-resolution microscope. Chromosome isolation and delivery is achieved by an opto-fluidic chip that is able to trap, visualise and lyse individual cells and separate metaphase chromosomes from cell lysate. Single chromosomes can be “hand-selected” and brought into focus of the Super-Resolution Correlative Tweezers Fluorescence Microscope (CTFM-SR3D) that is developed in CHROMAVISION. This instrument will for the first time enable 3D, super-resolution, real-time metaphase chromosome observation and manipulation studies under near-physiological conditions. The technique will push the boundaries of what is currently possible in microfluidics and super-resolution microscopy and combine these into a single powerful approach for chromosome studies. Furthermore, the platform will be applied in CHROMAVISION to address key challenges in clinical and fundamental chromosome research, potentially resulting in breakthrough discoveries. Better imaging and understanding of the chromosomal mechanisms will contribute to our knowledge of the etiology of human diseases and aid drug discovery. The platform will also have large clinical value, allowing identification and monitoring of e.g. cancer heterogeneity.
The main aim of the proposal is broken down into four R&D objectives:
1. To develop an integrated and intuitive Super-Resolution Correlative Tweezers Fluorescence Microscope (CTFM-SR3D) for chromosome imaging and manipulation that integrates lab-on-a-chip (LOC) microfluidics, multiple-trap optical tweezers and super-resolution fluorescence microscopy.
2. To develop and validate production ready prototypes of chromosome labs-on-a-chip for extraction, visualisation and actuation of metaphase chromosomes from single cells.
3. To develop methods for the quantitative study, in real time, of the 3D structure and dynamics of whole mitotic chromosomes extracted from healthy and diseased single cells
4. To apply the chromosome imaging and manipulation platform to address key questions in biomedical and fundamental chromosome research

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

WP1 - Development CTFM-SR3D
Task 1.1 Extend capabilities of current CTFM platform: larger range of optical manipulation + larger-range and faster confocal scanning (M1-M24): The range of the high resolution optical trap of the LUMICKS CTFM instrument (C-Trap) was extended by a factor of 1.66 (from 40 to 67 um) by using a custom mirror design with a 10 mrad range. Moreover, the Field-of-view of the bright-field camera used to detect bead positions was extended from 50x50 um to 100x100 um by modifying the optical path (shorter focal length tube lens, 2” mirrors and dichroics). While the three other traps have a range which is larger than the 100 x 100 um field of view. In addition, optical designs for various high speed scanner options have been made and evaluated. VU has supported all CTFM developments, is testing the overall system and is currently exploring implementation of a high-speed scanner.
Task 1.2 Develop and construct 3DSR-CTFM system with 3D STED (M1-M24): A Quadruple-trap optical tweezers that will serve as the basis of the CTFM system has been built and installed by Lumicks. Compatibility of The CTFM instrument with STED has been validated using a 1D-STED module designed, implemented and tested by Lumicks. A tip/tilt flowcell holder has been designed which allows the user to minimize aberrations affecting STED imaging. VU has evaluated, designed and built an SLM-based 3D-STED path and coupled it to the CTFM system. Software and hardware for full digital control of the 3D-STED point-spread-function was established and demonstrated. The required 1D, 2D and 3D STED PSFs have been obtained. Partial automation of aberration-correction using SLM has been implemented.
Task 1.3 Develop software for the 3DSR-CTFM capable of 3D imaging and handling of chromosomes (M1-M36):Lumicks software development for this project was split into two tasks: 1) for prototyping and to support the R&D of the physicists developing the instrument and workflows the current Labview software of the C-Trap has been extended with essential features and improved usability. This will enable more user friendly operation and will be invaluable for the complex workflows that are part of this project. Specific improvements include: SLM control for 3D Sted, improved control of trap powers, control of automated fluidics valves, improved template selection for bead tracking. 2) simultaneously a new software architecture based on Qt/C++ is under development which will enable us to deliver a much more stable, performent, integrated and polished user experience suitable for the end users (UCPH and UCL) during the field testing.
Task 1.4 Production-grade lab-on-a-chip (LOC) (M1-36): Tests were done to determine the suitable materials for the manufacturing of the SHIM for injection molding of the CHROMAVISION SHIM. We concluded that steel is the material to be used in the manufacturing of the SHIM. If the geometries change to sub 50 µm then Nickel Vanadium is to be used. Next, we made improvements to the injection molding process so a much higher surface quality can be achieved, even with a certain amount of deformation on top of the shim. Injection molding protocol named CHRM_20160505_OP8 is to be used in the injection molding process. Lid-bonding procedure: this process has been a challenging one. Test have been made using an ultra-sonic welding method, which can make an airtight seal around the channels, without the collapsing of the lid into the channels. This ultra-sonic welding is to be used in finalization of the SHIM

WP2 - Opto-fluidic Chromosome Chip Technology
Task 2.1 Integrate LOC platform with optical tweezers and STED microscopy (M1-M36): Lumicks shared chip designs with DTU for implementation as polymer chips. Tests of first polymer prototypes showed that the current quality is not good enough (see also task 1.4). Working with DTU on redesign and different bonding approaches. In parallel, Lumicks current chip layout is transferred to the three injection moulding tool platforms: Microfabricated Nickel shim, micromilled Al shim, milled and polished low-grain size tool steel. Injection moulding tool insert for Lumicks chip layout fabricated and tested. First Topas test chips moulded. Tests and process optimization for laser-manufacturing “energy directors” in new tool steel initiated. Feasibility confirmed.
Task 2.2 Develop a micro-fluidic platform to emulate chromatid segregation (M6-M24):We have re-established and improved the microfluidic platform and the protocol for preparing metaphase chromosomes from modified human cell lines. The platform was previously used to unfold DNA from a metaphase chromosome by proteolysis. We use this experiment to benchmark the improved set-up with previous results. DTU re-establish the microfluidic reactor experiment at DTU. We have successfully implemented an automation scheme that makes the experiment user-friendly so it can be implemented at UCPH. UCPH has improved the best-known protocol for making purified metaphase chromosomes from human cells. As a result of those improvements, several metaphase chromosomes can be unpacked by proteolysis in the same device in the course of a single experiment.
Task 2.3 Develop an opto-fluidic chip to obtain metaphase chromosomes from cells (M6-36): During the first period we tested our initial hypothesis that metaphase chromosomes can be released from a metaphase cell inside a microfluidic cell trap that DTU designed previously. In this device a cell is trapped in a microfluidic constriction and lysis buffer can be introduced to it thus the content of the cell is collected in an outlet channel. We tested several protocols for performing lysis and release of the chromosomes. The conclusion at this point is that the hydrodynamic trapping in a constriction is mechanically counteracting the release of the chromosomes from the cytoplasm of the cell even when the cell membrane is effectively lysed. Alternatively, we use sonication on-chip which introduce a mechanical strain on the cell to mimic vortexing that releases and disperses the chromosomes when prepared from many cells in a test tube (protocol used in task 2.2). We therefore could try to apply sonication to cells captured by optical trapping to improve releasing and separating chromosomes from single cells.
Task 2.4 Workflow for delivering all chromosomes from a single cell to the 3DSR-CTFM (M6- 24): In this task DTU made the preliminary steps to integrate a ‘mine field’ to collect metaphase chromosomes extracted from a cell on-chip. Our strategy is to create an array of optical traps inside a microfluidic channel by integrating plasmonic-based lenses in the microfluidic chip. So far we have shown that plasmonic lenses can be written in a metal metasurface. Those lenses can then focus green light. In parallel, we worked on trapping cells using an optical trap created by more conventional free space optics in order in order to gain experience about conditions necessary for optical trapping of cells.

WP3 - Quantitative analysis of chromosomes structure and dynamics
Task 3.1 Develop handles for metaphase chromosomes (M1-M6): A stable expression of the BirA*-CENPB or BirA*-TRF1 fusion protein is established. Currently, we are analyzing the effects of adding biotin to the cell culture medium. Our data show that this leads to specific biotinylation of proteins at the centromere (for CENPB) or the telomere (for TRF1). This then provides the potential to act as a ‘bead handle’ for each chromosome. We are conducting pilot experiments to ascertain whether streptavidin-coated beads can be made to attach to purified, ‘biotinylated’ chromosomes, and whether these beads are localized correctly to either the centromeres or the telomeres.
Task 3.2 Perform 3D STED imaging of stationary metaphase chromosomes (M7-M36): Metaphase chromosomes have been visualized using bright-field and fluorescence (wide-field) microscopy. We also showed that chromosomes respond to acoustic waves that push them towards nodes in the flow cell. This might be employed to increase the effective concentration of chromosomes. Currently, bead attachment to chromosomes has priority, since this will strongly promote both manipulation and visualization possibilities.
WP4 – Management, dissemination and exploitation
Task 4.1: Implementation of internal project MGT tools. M1 – M48: They have been established by all partners and the coordinator.
Task 4.2: Legal, ethical and gender issues and contractual management. M1 – M48: All these issues have been discussed and agreed upon.
Task 4.3: Implementation of internal progress reporting and monitoring procedures. M1 – M48: The procedures are deployed right now and this first reporting moment is a good test in order to improve our procedures.
Task 4.4: Coordination of the periodic technical and financial reporting. M1 – M48: An effective coordinator has established her role well. Communication between partners is effective.
Task 4.5: Planning, implementation and follow up of network meetings and EU project reviews M1 – M48: 2 network meetings have been organized. A method of collaboration has been established.
Task 4.6 Developing communication plan and corporate style; development, hosting and maintenance of a public website; development of dissemination material and activities M1-M48: A website has been launched and a flyer has been developed. Moreover, we developed a general communication plan.
Task 4.7 Development and execution of exploitation plan. M1-M48: An exploitation plan has been developed and uploaded as deliverable

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

At this first reporting stage we are still setting up the ground work of the project and hence we have not moved beyond the state of the art yet. Same is true for the impact of our action so far. We have communicated in our scientific community our plans which have been received with great interest but it is too early for societal implications of our work.

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