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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 2 - CHROMAVISION (Super-resolution visualisation and manipulation of metaphase chromosomes)

Teaser

Summary

CHROMAVISION aims to develop a pioneering chromosome imaging and manipulation platform to fuel future structural chromosome research. Chromosomal abnormalities are characteristic of many disorders such as cancer, impaired fertility, and neurological disorders. This platform will allow molecular biologists to isolate individual chromosomes from small tissue or cells and deliver them to a super-resolution microscope. Single chromosomes can be 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. Better imaging and understanding of the chromosomal mechanisms will contribute to our knowledge of the etiology of human diseases and aid drug discovery.
The overall objectives of the proposal are:
1. To develop an integrated and intuitive 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, visualization 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

WP1
Task 1.1: Most goals were already met during period 1. During the current period VU has tested a fast confocal scanner (Yanus II, FEI) on the LUMICKS CTFM platform. This task is finished.

Task 1.2: Most goals were met during period 1. 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 are obtained. This task is finished.

Task 1.3: LUMICKS developed the software for chromosome trapping and handling in Labview. A completely new control software for the platform is developed which yields improved performance and reliability. It further optimizes the user interface and allows automation of experiments. By integrating the coordinate systems of the traps, the bright field image and the confocal scanner the user can control a trap or select a region for super resolution scanning by simply clicking or dragging a selection in the camera image.

Task 1.4: Following initial test with Aluminum and (elelctromplated) Nickel Vanadium shims as reported for period 1, it was decided to proceed with the third option: A special tool set, manufactured in hard tool steel, suitable for manufacture of 10000+ parts has been finalized. Lid-bonding procedure: Ultra-sonic welding of plastic foil lids was tested, but failed to produce reproducible sealed microfluidic devices. Like for thermal bonding, the large footprint of the chip made it difficult to obtain good results. Solvent assisted bonding is currently under test with positive results.

Task 1.5: We succeeded to develop a workflow to handle single purified metaphase chromosomes that feeds into the further technical hardware and software development. (see also task 3.2).
WP2
Task 2.1: LUMICKS has demonstrated reliable and stable trapping of beads in all polymer chips obtained from DTU. DTU produced chips by molding 1mm thick TOPAS tops with the microfluidic channels. These were covered with 150 um thick lids made by flattening TOPAS foil. The devices were tight at low pressure. At high pressures liquid leaked. This was expected since the mold used for injection molding was originally designed for bonding via ultrasonic welding. We are currently developing a new version of the device that is better suited for solvent-assisted bonding.

Task 2.2: The micro-fluidic platform works and we are using our device to investigate ways to expand metaphase chromosomes besides proteolysis. We used a high-salt solution to induce the chromatin expansion. This is the first demonstration in our microfluidic reactor without tethering to surfaces.

Task 2.3: During period 2 we dropped our first hypothesis of using a chemical based lysis process. Instead we worked on using on-chip sonication to include vortexing in the microfluidic chip and disrupt the metaphase cells. We will investigate new device design to maximize the shear experienced with cells. We will also use other cells that may be more fragile to start with.

Task 2.4: DTU made the preliminary steps to integrate a â€˜mine fieldâ€™ to collect metaphase chromosomes extracted from a cell on-chip. Our strategy is to create an array of optical traps by integrating plasmonic-based lenses in the microfluidic chip. DTU has developed a method for laser printing of flat optical components in prefabricated metasurfaces which can be manufactured by production-grade methods and laser reshaping to provide control over optical metasurface functionality.

Task2.2: During period 2, we have adopted the approach of purifying recombinant active human separase from E. coli to cleave centromeric cohesion in vitro, enabling the sister chromatids to come apart. Two constructs were made, but neither of them produced an active form of separase. We are currently making new constructs for this purpose.

WP3
Task 3.1: We have established HT1080 cells with stable expression

Final results

At this reporting stage we are deep in the development of our research plan, our successes routinely manipulating single metaphase chromosomes can be considered beyond the state of the art. It is still too early to judge the potential impact other than what we described in the proposal. But when communicate in our scientific community our results are received with great interest.