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Chromatin organization PROfiling with high-throughput super-resolution microscopy on a CHIP

Periodic Reporting for period 2 - PROCHIP (Chromatin organization PROfiling with high-throughput super-resolution microscopy on a CHIP)

Reporting period: 2019-09-01 to 2022-02-28

The aim of the PROCHIP project was to study cellular heterogeneity in large populations, allowing the classification of isolated cancer samples based on their chromatin architecture. To this end, we aimed at developing a super-resolution microscope with high throughput capabilities, able to acquire thousands of cellular samples.
We exploited laser writing technology, Femtosecond Laser Irradiation followed by Chemical Etching (FLICE) to develop a light sheet fluorescence microscope (LSFM) on a chip, equipped with structured illumination microscopy (SIM). FLICE allows fabricating optofluidic components as waveguides, microchannels and lenses in a glass substrate. By combining these components, a mm-scaled lab-on-chip with integrated illumination path and sample delivery has been realized.
In details the objectives of the project were:
1) development of a miniaturized three-dimensional fluidic network integrated on a glass chip and its relative pumping system for automatic sample scanning under a microscope.
2) development of a super-resolution microscope, whose illumination and sample scanning components are integrated in a lab-on-chip (super-resolution microscope on-chip).
3) identification of the protocols for isolation of primary and metastatic tumor cells suitable for high-throughput imaging and data analysis at super-resolution based on the image simulator.
4) assessment of cancer cell heterogeneity by querying chromatin domains as functional biomarkers.

We realized different prototypes of microscopes on chip, in which the sample flowing in a microchannel is optically sectioned by a uniform or by a patterned light sheet, capable of automatic sample scanning at high throughput.
The first challenge was the achievement of a stable flow control, to scan the samples in an automated manner. The second was to realize a patterned light sheet within a microchannel for the optical sectioning of a flowing sample with structured light to enhance the spatial resolution. A third challenge concerned the realization of software for setup simulation and design, for remote controlling of the system during the measurement acquisition and for the image reconstruction and analysis.
In conclusion, although phenotypic profiling of chromatin domain has not been completed, all the means necessary for its successful realization have been developed, ranging from the development of protocols for sample preparation and analysis to the realization of an optofluidic microscope on chip capable of high throughput 3D imaging of fluorescent single cells with enhanced resolution.
The project was structured in six work packages, four of which concerning research and development activities.
WP1 was dedicated to identifying the protocols for sample preparation by UNITN (isolation of cancer cells from primary and metastatic organs and sample labelling), for sample handling by ELVESYS and CNR (insertion and controlled motion of the sample within the chip). An open-source software which assembles most fluorescent microscopy simulators was developed by UA and INSA Lyon and it’s now available online.
WP2 aimed to set the basis for high-throughput LSFM in a fluidic chip, by CNR. In Year 1 a milestone concerning a closed-loop fluidic system for single cell movement by ELVESYS and CNR has been achieved.
WP3 was devoted to design and build LSFM on chip with patterned light. In Year1 the design of the integrated platform for superresolution analysis was stated by IMPERIAL and CNR. A delay in the prototype realization occurred because of the pandemic, anyway CNR achieved the implementation of an integrated pattern generator that is used to perform SIM on chip.
WP4 aimed at performing chromatin profiling in cancer cells. IMPERIAL partner acquired images of samples produced by UNITN with conventional SIM and with a new SIM architecture developed on purpose. CNR achieved high throughput automatic scanning of cancer cells produced by UNITN in standard resolution LSFM on chip and preliminary measurements on LSFM-SIM on chip.
WP5 and WP6 concerned respectively the dissemination and management of the project.

The main achievements of the PROCHIP project can be summarized in the following points:
- A new method to realize SIM based on the interference of three coherent beams (called HexSIM) was proposed and implemented.
- A pipeline for the identification, quantification and measuring of chromatin domains has been validated by isolating cancer cells from primary tumors and metastatic lesions and labelling chromatin domains to determine their distribution and organization
- Different prototypes of microscopes on chip have been realized and validated for automatic LSFM, SIM with standard and superresolved image reconstruction
- A software for cell phenotyping (MicroVIP) has been developed and deployed. The tool allows realistic high-resolution simulation of large microscopy images, with annotations required for machine learning.
- New routines for instruments control, software for demodulation and SIM reconstruction have been developed and made available online.

PROCHIP results were disseminated to the scientific community through >25 presentations (contributed and invited), 15 publications in peer-reviewed journals and conference proceedings, about 10 events with large participation of general public. Two patent applications were filed during the project duration.
The PROCHIP approach promised to go well beyond the state of the art in different field: (i) photonics, (ii) computer sciences and (iii) cancer research.
i) We increased the capabilities of LSFM providing a platform for high-throughput 3D imaging of cells with high resolution. Structured illumination on the cells flowing in the channel has been demonstrated, paving the way to high throughput superresolution microscopy. Original results have been obtained in the machining of borosilicate glass and in the smoothening of fused silica etched surfaces. The results impact the fabrication of glass components by femtosecond laser writing, an industrial sector that is already experiencing an increase in the investments. The realized prototypes will be used for the generation of patterned light illumination systems to be used in different microscopy applications.
ii) An image simulator (MicroVIP) has been developed for research and development in bioimaging applications. It can be used for virtual instrumentation by realizing low cost and fast in silico tests to determine the best tools and settings before a real microscopy experiment; for machine learning applications such as deep learning based super-resolution or data augmentation as it enables fast and inexpensive realization of a 3D microscopy images database. The software can be used in the virtual imaging platform VIP (
iii) Significant results have not yet been achieved in the unravelling of epigenetic effects since the integrated devices for high throughput measurements have been validated with specific cell populations. Anyway, results on the procedures needed to prepare the samples of interest have been obtained; superesolved images with the newly proposed hex SIM has been acquired and simulations and evaluations of the parameters necessary for imaging in microchannels have been conducted, facilitating future microfluidic imaging analysis.
PROCHIP roadmap; steps towards the goal: chromatin profiling with superresolution microscopy on chip
Rendering of the Structured Light Sheet Microscope on chip realized within the PROCHIP project