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Plasmonic Electronically Addressable super-Resolution: Accelerating the in-depth understanding of biomedical processes at the nanoscale via a novel real-time, optical limit-breaking imaging technology

Periodic Reporting for period 1 - PEAR (Plasmonic Electronically Addressable super-Resolution: Accelerating the in-depth understanding of biomedical processes at the nanoscale via a novel real-time, optical limit-breaking imaging technology)

Reporting period: 2019-06-01 to 2019-10-31

What is the problem/issue being addressed?

As the routinely used method to identify biological processes (optical microscopy) is incapable of imaging nano-scale objects, it is extremely limited in unraveling mechanisms at this scale. However, as emphasized by the most recent Nobel Prize (medicine 2019), understanding the detailed mechanisms
e.g. in the human body is key to develop new therapies, capable of effectively fighting diseases such as cancer. Our technology enables researchers to visualize those mechanisms in real-time, increasing our understanding of biological pathways. As a result, this will enable us in the future to manipulate such
pathways which in addition to improved diagnostics will result in new therapies.

Current state of the art:
In general, microscopy and in particular fluorescence microscopy is diffraction limited as a result of the wave nature of light. Mathematically speaking, imaging in the far field does not preserve the high frequency
spatial components in the transmitted light and therefore is losing information. Abbe calculated the diffraction limit (subject to assumptions regarding the criteria for optical resolution) to be of the order of 200 nm for visible light.
This barrier remained in place for roughly a century (despite suggestions to overcome this limit by E.H. Synge in the 1930s), and practically barred optical microscopy and related techniques to identify objects at the nanoscale.
In 2014, the Nobel prize for chemistry was awarded to three researchers (S. Hell, E. Betzig, and E. Moerner) for the innovation of novel methods which overcome the diffraction limit namely the deterministic STED, and the stochastic PALM/STORM.

Advancing the current state of the art:
In contrast, PEAR offers a highly innovative solution that goes beyond the current state of the art. It will enable real-time nano imaging of cancer cells and drug interactions, which is currently unavailable but demanded by cancer researchers as a key technology:
As our innovation is already providing spatial resolutions below most other super resolution techniques at its current stage, but as its ultimate resolution scales with the size of the sub-structures embedded on the optical chip, combined with the ongoing improvements in lithography, it is envisagedthat PEAR Nanoscopy can be scaled down routinely to below 10 nm spatial resolution. This is unique for a deterministic, label-free imaging technology.

Why is it important for society?
This novel disruptive imaging technology will be instrumental in early detection and development of next generation therapies for cancer.
Cancer is the biggest killer in Europe, accounting for e.g. over 9,000 deaths (~30% of deaths – one person every hour) every year in Ireland alone. Globally, cancer was responsible for an estimated 9.6 million deaths in 2018.

What are the overall objectives?
We present an entirely novel and IP protected method enabling video-rate nano-scale imaging. PEAR is an acronym and stands for Plasmonic Electronic Addressable Super-Resolution.
Successfully completed work to date:

1. FDTD Simulations have proven localized field changes,
2. Created initial setup to demonstrate “lock-in” onto the modulation of pixel,
3. Incorporated such pixel into XY-piezo stage,
4. Demonstrated that scanning the pixel does yield imaging,
5. Proof of concept on an experimental setup: thermal lock-in signal, physical setup for nanoscopy using an inverted microscope.
6. Preliminary setup with macro pixel, optimization of pixel size and scanning
7. Refined the pixel size,
8. Successful design of multi-pixel arrays. Reduction of signal to noise ratio and further acceleration of acquisition time.
9. Iterative use of FDTD computer simulations which had shown us the working principle and the possibility to achieve resolution beyond the diffraction limit of light. These
simulations were used during the experimental stage to configure the setup to the optimum parameters including laser wavelength and incident angle.
10. Improved signal-to-noise ratio.
11. Incorporation: sample preparation aspects
12. Commercialization
Our results show deterministic imaging of biologically highly relevant samples in comparison to the image quality a high numerical aperture microscope can deliver.
The differences are striking as the PEAR image shows a much greater level of detail with an order of magnitude higher spatial resolution.
Due to the confinement of the electric field around the pixels, the resolution of this method is directly dependent on the physical size of the elements. The limit of our imaging method is now only reliant on
nanolithography which is highly advanced. An increase in resolution is straightforward since nanotechnology is currently at the scale of 7 nm. Our imaging solution can be directly applied to research, such as
investigations into cancer, that have until now been held back by the inability to view cells and processes on this scale, and in real time, both of which is crucial in the design of novel therapies.
Our innovation is providing spatial resolutions below 21 nm at its current stage, which is one order of magnitude beyond the diffraction limit of light.

Demonstrated advantages of PEAR Imaging beyond the current state of the art:

Our technology has the following advantages:
• Spatial resolutions far beyond the diffraction limit – currently at 21 nm
• Deterministic
• Label-free (labels can be used for enhanced contrast but are not strictly required)
• In-vitro capabilities (biocompatible)
• Easy integration into existing microscopes
• Fast (video rate or higher) imaging, parallel read-out instead of scanning
• Exchangeable optical chip

IP protected invention
The Invention is already protected via a patent filing and has recently been progressed to the PCT stage:
D. Zerulla, P34145WO “Addressable Plasmonic Arrays”, European Patent Application, filed 21. December 2017; progressed to PCT stage December 2018.
Further patents have been recently filed 06/2019 and are currently planned. NovaUCD is providing further support to ensure that the invention is well protected and the potential leveraged.

Socio-economic impact and the wider societal implications of the project:

(a) Improved treatment: Our PEAR microscope system provides a tool for observing and understanding
the mechanism of new drug treatments (e.g. nanomedicine).
(b) Earlier detection: PEAR microscope system can aid this through high resolution for rapid point of care diagnostic.

The next big demand in microscopy with a major societal impact will come from compact, robust and affordable optical systems. This will make high-resolution microscopy highly accessible, enabling both
ultra-high resolution to aid researchers in developing improved personalized therapies and secondly (our long term objective) enable point-of-care, by facilitating early diagnosis for cancer and other
genomic disorders. This is the societal impact mission of the PEAR innovation.