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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - MUSICAL (Chip-based MUSICAL nanoscopy for imaging endocytosis pathways of phage viruses in liver sinusoidal endothelial cells)

Teaser

Summary

The present state-of-the-art of optical nanoscopy lacks to provide high optical resolution (~50 nm) at high temporal resolution (~1 Hz) over large field of view (> 500 X 500 Î¼m2) in live-cell friendly imaging conditions, such as without special buffer/fluorophores and with minimum photo-toxicity.

The development of high throughput nanoscopy using integrated optics and nanoscopy algorithms will allow for high throughput screening of biological samples and pathological samples at significantly lower costs, smaller time scales, and simpler infrastructure needs than electron microscopy. This will enable easier diagnostics and drug development. Ability to image living cells and cell colonies will help in studying disease onset and progression and effect of drugs at the functional level (i.e. cell organelles) inside living cells.

In this project, we have delivered the overall objective of achieving ~50 nm optical resolution at small temporal scales (~ seconds) in photochemical environment which is physiologically conducive for in-vivo bio-imaging applications. A secondary objective that was delivered was the exploitation of the illumination patterns supported by photonic chip by computational nanoscopy algorithm MUSICAL to achieve large field-of-view super-resolution live cell imaging. A tertiary objective of training the experienced researcher (ER) for transitioning into faculty position was achieved successfully after the ERC starting grant application 3D-nanoMorph was granted to the ER in 2018. The ERC starting grant started after the completion of this project.

Work performed

The proof-of-concept of MUSICAL on chip (computational nanoscopy algorithm for exploiting photonic chip illumination diversity) was demonstrated. The photonic chip-based illumination system was automated and optimized for stability of illumination, automatic switching of illumination, and better illumination efficiency such that it can be integrated with MUSICAL. MUSICAL on chip was tested for fixed cells and tissues, compared and benchmarked with STORM, and successfully tested from correlative light and electron microscopy. Scalable resolution and FOV was demonstrated. Concept of using illumination variation for super-resolution was further developed into a technically rigorous concept. ~50 nm resolution with temporal resolution of the order of tens of seconds was achieved using MUSICAL on chip. Further speed up is possible by superior instrumentation and will lead to imaging of the order of 2-5 seconds. Large FOV of upto 1.5 mm x 1.5 mm region was demonstrated. MUSICAL was tested for a large variety of different biological samples on conventional microscopes and using wide variety of dyes and proteins as well as autofluorescence. MUSICAL\'s versatility for a wide range of biological systems including living cells was proven. Therefore, scientific achievements beyond the originally proposed objectives were achieved.

ERC starting grant application was submitted by ER in October 2017. The funding was successful. ER supervised or co-supervised 2 master students (graduated) and 1 PhD student (onging) as a part of Marie Curie project, worked with a team of about 10 people for accomplishing the multi-disciplinary scientific tasks listed above. ER developed knowledge in integrated optics and live cell imaging to understand the basic workings, strengths, and concerns while working with them. They feature as important aspects in the ERC starting grant projects as well as in other projects/proposals which the ER is part of. ER now boasts of extensive collaboration across Europe, UK and in fields spanning technology, biology, mathematics, computer science, etc. ER was enrolled into Digital Life Excellence program and UiT\'s Aurora Outstanding program for mentoring young research leaders. Therefore, all the training objectives of the project were met.

MUSICAL\'s source codes and data on the popular imageJ platform were released as plug-in with a simple user interface for the ease of use of researchers. Project webpage and Youtube videos were created and disseminated. See links:
https://www.3dnanoscopy.uit.no
https://github.com/sebsacuna/jmusical
https://sites.google.com/site/uthkrishth/musical
https://youtu.be/deCu_tXoloY
http://youtu.be/CsJHqSQb11E
The work was presented in 12 national and international conference presentations. Two journal articles have been published in Scientific Reports. Few more journal articles are being prepared. Invention disclosure on high computational throughput of MUSICAL has been filed and patent application is being prepared. The training and networking opportunities were exploited for significant enhancement in the collaborative network in the ER.

Final results

Super-resolution with low photo-toxicity can be achieved by integrating MUSICAL with photonic chip and exploiting the illumination diversity instead of photokinetics of fluorophores. It can be used for imaging living cells, cell systems, and tissues. Resolution and FOV scaling can be achieved easily now, allowing for both high throughput screening of biological samples, and selective nano-scale imaging on the same imaging system. This will help in catering to clinical pathology, drug development, and large scale population studies. In addition to the open access ImageJ plugin, one to one dissemination of MUSICAL has been conducted in 5+ research labs all over the world. These labs are exploring the utility of MUSICAL for deriving insights and therapy solution for cardiovascular, neurological, and cancer diseases. It is expected that the freely available ImageJ plugin will bring nanoscopy to the doorsteps of every small lab having a simple microscope. Additionally, the invention disclosure on high computation throughput solution is being prepared the patent application. It will, through commercialization efforts, lead to highly optimized solutions for clinical and pharmaceutical applications.