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Focus on Advancing Spatial and Temporal Resolution of 3D Structured Illumination Microscopy

Periodic Reporting for period 1 - FASTR 3D SIM (Focus on Advancing Spatial and Temporal Resolution of 3D Structured Illumination Microscopy)

Reporting period: 2017-11-06 to 2019-11-05

Fluorescence microscopy is an important tool in biological and biomedical research, as it allows researches to visualize structures and dynamics on a cellular and sub-cellular level.

Two common limitations in the study of inner- and intra-cellular dynamics are the resolution and the speed that can be achieved. Light diffraction limits how fine the structures are that a microscope can resolve, and technical implementation limits how fast dynamics can be observed.

This project aimed to improve both the speed and the resolution with which we can observe those dynamics. More specifically, new technical solutions (implementing faster microscopes based on novel optical elements) and the improvement and combination of existing microscope methods, are used to achieve this goal.

The successful implementation will provide vital tools for biological and biomedical research, especially those fields aiming to observe fast dynamics at highest resolutions. For example, the visualization of mitochondrial dynamics require both the resolution and speed we aim to provide.
We have constructed a fast structured illumination microscope that can acquire volume information (3D images) at very high speeds. By using a novel optical element, multiple different slices of a sample can be projected onto two scientific cameras, and imaged at the same time, without the need to refocus the sample. This speeds up data acquisition without degrading image quality.

We have also completed an on-the-fly data processing system for SIM microscopes. Typically, the data processing is performed off-line after the experiment is completed. With our systems, researches now have access to reconstructed, super-resolved image data while the experiment is still running. This allows to adjust experimental conditions or react to the biological dynamics observed without waiting for a time-consuming post-processing step.

Both microscope system were demonstrated by imaging mitochondrial dynamics, a prime target for this high-speed imaging approach. We also demonstrated their use for other imaging tasks, for example the fast readout of DNA barcoding samples.

The systems developed have been made available to the scientific community in open-access publications, their blue-prints and software components are freely available under open licenses. A new device developed as part of the research is currently being prepared for a patent application.
A novel, high-speed structured illumination microscope (SIM) was constructed, allowing for increased 3D imaging of fast intra- and inner-cell dynamics.

A combination of a bespoke SIM microscope and a GPU-accelerated, high-speed data processing platform was implemented, which for the first time allows the users of such a microscope to view the reconstructed, super-resolved data in real time.

Further devices developed during the course of the project are being evaluated for a patent application. Method development continues for an improvement of the resolution achieved in SIM imaging.

The ability to observe dynamics at high speeds provides a new tool to biological and bio-medical research. A first application, carried out at the KU Leuven together with collaboration partners, will be to image mitochondrial dynamics.
Video-rate, multi-color SIM system blueprint (DOI: 10.1038/s41467-019-12165-x)
On-the-fly data processing of SIM images (DOI: 10.1038/s41467-019-12165-x)