Integrated high-resolution on-chip structured illumination microscopy

Project Information

IROCSIM

Grant agreement ID: 805222

DOI

10.3030/805222

Project closed

EC signature date 27 September 2018

Start date 1 January 2019

End date 31 December 2023

Funded under

• EXCELLENT SCIENCE - European Research Council (ERC)

Total cost

€ 1 499 625,00

EU contribution

€ 1 499 625,00

Coordinated by

INTERUNIVERSITAIR MICRO-ELECTRONICA CENTRUM
Belgium

Periodic Reporting for period 4 - IROCSIM (Integrated high-resolution on-chip structured illumination microscopy)

Reporting period: 2023-07-01 to 2023-12-31

Summary of the context and overall objectives of the project

Fluorescent microscopy is indispensable in biology and medicine and has fuelled many breakthroughs in a wide set of sub-domains.
Recently the world of microscopy has witnessed a true revolution in terms of increased resolution of fluorescent imaging techniques. To break the intrinsic diffraction limit of the conventional microscope, several advanced super-resolution techniques were developed. High resolution microscopy is also responsible for the spectacular cost reduction of DNA sequencing during the last decade.
Yet, these techniques remain largely locked-up in specialized laboratories as they require bulky, expensive instrumentation and highly skilled operators.
The next big push in microscopy with a large societal impact will come from extremely compact and robust optical systems that will make high-resolution (fluorescence) microscopy highly accessible.
IROCSIM facilitates this next breakthrough by introducing an innovative high-resolution, compact microscopy platform that provides a fully integrated lens-less fluorescence imaging solution with sub-pixel resolution, and that is based entirely on an intimate marriage of active on-chip photonics and CMOS image sensors.

The resulting platform will enable high-resolution, fast, robust, zero-maintenance, and inexpensive microscopy with applications reaching from cellomics to DNA sequencing, proteomics, and highly parallelized optical biosensors.

The IROCSIM project team worked on the key building blocks of this technology. We developed and validated algorithms to design photonics circuits based on the required structured illumination (SI), image reconstruction algorithms, integrated optical phase modulators and beam splitters, excitation filter, and a process flow for deposition of active components, waveguides, and interference filters on imager wafers.

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

The main results obtained in the development of the IROCSIM imaging technology are:

- A theoretical framework describing how to generate a sparse optical lattice illumination in a photonic integrated circuit (PIC) with a minimal number of input waveguides. Experimental validation of our optical lattice method was initially obtained using free space optics. Hexagonal, square, and quasi-lattices can be generated and scanned. Published in [1,2], presented at [3,4]. We also explored inverse design methods for the generation of periodic optical patterns in PICs with reduced footprint [5,6].
- Development of an efficient thermo-optic phase shifter and switch matrix in the visible range on a Silicon Nitride platform and a scalable multichannel current source driver. These components are essential for the practical implementation of the interference patterns and to scan them. [7-10].
- Experimental demonstration of structured illumination microscopy (SIM) using our active integrated photonics platform for the generation and modulation of the sinusoidal structured illumination patterns in combination with our custom developed reconstruction algorithm. A resolution of 110 nm was achieved. [11-14]
- A custom fluorescence excitation filter that is placed between the imager and the photonic circuit was developed. This filter rejects the excitation light over a large angular range so that only the fluorescence emission reaches the imager.
- A full fabrication process flow for the monolithic integration of an active photonic integrated circuit with an excitation filter on top of a front-side illuminated (FSI) CMOS imager. [13,14]
- Packaged functional proof-of-concept chips on FSI imager. Chip performance evaluated using fluorescent nanospheres and uniform illumination: image sensor functional; filter performance allows sufficient background suppression resulting in signal-to-noise (SNR) of 30; phase modulators functional; photonic components characterized. [13,14]
- A system model for predicting point-spread function, SNR, resolution. All chip components are included in this model that helps to guide future system design and optimization, and evaluate reconstruction algorithms.
- A compact beam expander with the potential to enable a large field of view. A publication is accepted [15].
- A demo setup to illustrate our on-chip structured illumination microscopy technology at exhibitions. The project team presented this live demo at the tech exhibition of the imec Future summits 2022 in Antwerp.

1) D. Kouznetsov, et al. Phys Rev Lett 125, (2020).
2) D. Kouznetsov, et al. Phys Rev A 105, (2022).
3) D. Kouznetsov et al. Biophotonics and Imaging Graduate Summer School (BIGGS, 2020)
4) D. Kouznetsov, et al. Complex Light and Optical Forces XV vol. 11701 1170117 (SPIE Photonics West, 2021).
5) D. Kouznetsov, et al. Optics Express 30(7), (2022).
6) D. Kouznetsov, et al. Proceedings Optica Advanced Photonics Congress 2022 IW5B.4 (2022)
7) Q. Deng, et al. Integrated Photonics Platforms: Fundamental Research, Manufacturing and Applications vol. 11364 52 (SPIE Photonics Europe, 2020).
8) Q. Deng, et al. Asia Communications and Photonics Conference/International Conference on Information Photonics and Optical Communications 2020 (ACP/IPOC) T4D.2 (2020).
9) Q. Deng, et al. China Semiconductor Technology International Conference (CSTIC) (2020)
10) N. Verellen, et al. European conference on integrated optics (ECIO, 2020) & O. Arisev, et al. Biophotonics and Imaging Graduate Summer School (BIGGS, 2020)
11) O. Arisev et al. ICON Europe 2021 – 3rd international conference on nanoscopy (2021)
12) O. Arisev, et al. Proceedings Optica Advanced Photonics Congress 2022 ITu1B.5 (2022).
13) N. Verellen, et al. 4th International Conference on Optics Photonics and Lasers (2023)
14) P. Van Dorpe, et al. SPIE Photonics West (2024)
15) D. Kouznetsov, et al. Journal of Optics (accepted 2024)

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)

Triggered by the need for arrays of individually resolvable excitation foci to achieve lens-free sub-pixel resolution, coherent lattice theory describes a unique approach to design structured interference patterns. Our approach to generate large-periodicity lattices, presents a method for efficient computation of coherent lattices, successfully covering all periodic and quasi-periodic lattices. Moiré theory and prime number factorization are the foundation of the proposed method. Moreover, our approach is not limited to light waves and therefore opens the door to new ways of studying interference patterns based on algebraic number theory. We experimentally verified key optical coherent lattices.

We were able to demonstrate a fully integrated photonic circuit for the generation of structured illumination in slab waveguides and use it to demonstrate an on-chip variant of TIRF (total internal reflection fluorescence) based classical SIM (structured illumination microscopy) using sinusoidal patterns. We were able to experimentally demonstrate 110 nm optical resolution, representing a 3-fold resolution enhancement over conventional wide-field microscopy with the same field of view. With this we went well-beyond the state-of-the-art in terms of integration level, image quality, and resolution.

Chips that allow to experimentally demonstrate the on-chip optical lattice illumination and lens-free high-resolution read-out with the monolithically integrated CMOS image sensor have been designed, fabricated, packaged, and components characterized. With these chips, we are continuing our efforts to validate our innovative lens-free fluorescence imaging concept.

Chip-scale microscopy for high-throughput fluorescence imaging