NANO-scale VIsualization to understand Bacterial virulence and invasiveness - based on fluorescence NANOscopy and VIBrational microscopy

Project Information

NanoVIB

Grant agreement ID: 101017180

DOI

10.3030/101017180

Project closed

EC signature date 8 December 2020

Start date 1 January 2021

End date 31 December 2025

Funded under

• INDUSTRIAL LEADERSHIP - Leadership in enabling and industrial technologies - Information and Communication Technologies (ICT)

Total cost

€ 5 635 530,00

EU contribution

€ 5 635 529,00

Coordinated by

KUNGLIGA TEKNISKA HOEGSKOLAN
Sweden

Periodic Reporting for period 2 - NanoVIB (NANO-scale VIsualization to understand Bacterial virulence and invasiveness - based on fluorescence NANOscopy and VIBrational microscopy)

Reporting period: 2022-07-01 to 2024-06-30

Summary of the context and overall objectives of the project

In this project, we are developing a microscopy platform for investigation of cells in great detail and to demonstrate its capabilities in biomedical research, for better understanding of the origins of cellular diseases. More specifically, the objectives are:

• construct prototypes of a next-generation fluorescence super-resolution microscopy platform, based on the MINFLUX concept, for operation in the near-infrared (NIR) wavelength range, offering one order of magnitude higher spatial resolution than current state-of-the-art super-resolution microscopes in this wavelength range.
• develop new single-photon detector arrays with enhanced sensitivity in this wavelength range.
• develop a pulsed, narrow-linewidth, multi-line laser for cellular imaging based on label-free inelastic light scattering.
• integrate the developed lasers and detector arrays into the prototypes of the super-resolution microscopy platform, offering a broadened wavelength range for imaging, faster image acquisition, lower background, and allowing correlative super-resolution fluorescence and label-free imaging of cells.
• as a lead demonstration for this platform, resolve nanometer scale localization patterns of specific proteins in bacteria and host cells, providing overlaid morphological and chemical images of the bacteria, representing key information on the mechanisms underlying virulence and invasiveness of the bacteria.

With the developed microscope platform and this lead application, we expect to take a decisive step towards better diagnostics, effective treatments and prevention of serious bacterial infections causing significant morbidity and mortality world-wide. The ability to resolve nano-scale localization patterns in cells, correlated to their morphology and sub-cellular environments, we expect will also open new means to understand, diagnose and prevent other diseases. To promote this development and such applications, we will offer access to a developed microscope system to biomedical researchers outside of the project. Moreover, since the three companies involved in the project will make their developments commercially available, a yet broader group of researchers in the biomedical field will have access to this technology in the future.
We thus expect that this project will contribute to maintaining a leading role for the photonics industry in Europe, at the same time new means to detect and understand cellular diseases, of large societal relevance, in Europe and worldwide.

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

Within the two (out of three) project periods, work has proceeded as expected, on all the major components needed to reach the goals:
Prototypes of the MINFLUX-based, next-generation fluorescence super-resolution microscopy platform are on place, operating in the near-infrared (NIR) wavelength range, offering one order of magnitude higher spatial resolution than current state-of-the-art super-resolution microscopes in the same wavelength range.
New single photon avalanche detector (SPAD) arrays have been developed with enhanced sensitivity in the NIR, and protocols for integration of the SPAD arrays into the MINFLUX platform have been established, offering additional means to enhance resolution and supress background.

A pulsed, narrow-linewidth, multi-line laser prototype has been assembled for label-free cellular imaging. After software development and successful optical performance tests, fulfilling requirements of fast spectral and pulse-width tuning, laser prototypes have been integrated into the two microscope prototype systems in the project.
The operation of the microscope prototype platform has been extended beyond the visible, into the NIR range. Integration of SPAD arrays into the platform prototypes has been accomplished and the pulsed, narrow-linewidth, multi-line laser prototypes have been installed into the microscope platforms and demonstrated for label-free morphological and chemical imaging of bacteria and human cells.

To make NIR-MINFLUX imaging possible, a range of NIR fluorophores have been investigated with respect to their blinking/switching properties, photostability and brightness, all critical parameters for super-resolution microscopy. We have then established excitation, imaging and sample conditions allowing NIR MINFLUX imaging of proteins on bacteria and human cells with nanometer localization precision.

With the MINFLUX platform, we can now image spatial localization patterns of proteins in bacteria and host cells with a few nanometers resolution, both in the visible and in the NIR. Harnessed with a pulsed, narrow-linewidth, multi-line laser, we can also perform label-free morphological and chemical imaging of bacteria and host cells. With this platform at hand, we have this far identified new localization patterns for at least two proteins coupled to bacterial disease and expect to present overlaid morphological and chemical images within the next months. Next, by further addressing this lead application, and by offering access to one of the developed microscope systems to biomedical researchers outside of the project, we will pave the way for new means to understand, diagnose and prevent cellular diseases. At the same time, these efforts will provide a test bed for the prototypes developed in the project and facilitate their way to market.

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)

In this interdisciplinary project, we are prototyping a next-generation super-resolution microscope system. This next-generation super-resolution microscopy offers an additional order of magnitude higher resolution than current state-of-the-art super-resolution microscopy techniques. Implementations that are currently available to researchers, however, are restricted to the visible wavelength range and use point detectors. Allowing the use of NIR light, using the noise suppression capabilities offered by array detection and providing cellular context through label-free imaging will make it possible to reveal yet unresolved, intricate, detailed molecular mechanisms underlying inter- and intracellular processes and disease.
As a lead application, we are in the process of demonstrating these unique capabilities to understand virulence and invasiveness of pathogenic bacteria, more precisely pneumococci. Pneumococcal infections are a major cause of morbitity and mortality worldwide, and it is highly needed to understand these mechanisms in a better way. By concerted development of laser and detector technologies, microscopy devices and image acquisition procedures, we will be able to retrieve information, which is not within reach by any other microscopic or photonic techniques. We will demonstrate how cellular nanoscale protein localization patterns can be resolved, which will not only help us revealing mechanisms of bacterial disease, but which will also likely be of large relevance for many other diseases. Thus, the microscope system prototype will prove its broad applicability in biomedical research, as a tool to understand intra- and intercellular processes and the cellular origin of diseases. From a methodological point of view, this project will support the development of cutting-edge microscopy, laser and detector technologies, which will find valuable use not only for the microscope technology to be developed in this project, but for a much broader range of applications. The project thereby will help European photonics industry and research to maintaining and further strengthening its leading position.

Stimulated emission depletion (STED) super-resolution image of surface proteins on pneumococci.