Periodic Reporting for period 1 - NanoVIB (NANO-scale VIsualization to understand Bacterial virulence and invasiveness - based on fluorescence NANOscopy and VIBrational microscopy)
Reporting period: 2021-01-01 to 2022-06-30
In this project, we will develop a microscopy platform for investigation of cells in great detail and demonstrate its capabilities in biomedical research, for better understanding of the origins of cellular diseases. More specifically, we will:
• 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. We also expect that the ability to resolve nano-scale localization patterns in cells, correlated to their morphology and sub-cellular environments, will open new means to understand, diagnose and prevent other diseases. To promote this development and such applications, we will offer access to one of the developed microscope systems 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.
• 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. We also expect that the ability to resolve nano-scale localization patterns in cells, correlated to their morphology and sub-cellular environments, will open new means to understand, diagnose and prevent other diseases. To promote this development and such applications, we will offer access to one of the developed microscope systems 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.
In the first period of the project (first 18 months) work has proceeded on all the major components needed to reach the aforementioned goals:
We have designed prototypes of the next-generation fluorescence super-resolution microscopy platform, based on the MINFLUX concept, for operation in the near-infrared (NIR) wavelength range and offering one order of magnitude higher spatial resolution than current state-of-the-art super-resolution microscopes operating in the same wavelength range.
The development of new single photon avalanche detector (SPAD) arrays with enhanced sensitivity in the NIR are likewise making significant progress. Dedicated detection electronics have been modified, and successfully integrated into the microscope prototype platform. SPAD array chips with enhanced sensitivity have been designed for later integration.
A suitable setup for a pulsed, narrow-linewidth, multi-line laser for label-free cellular imaging has been identified. Following optical layout calculations, and tests and validation of concepts, the mechanical design and electronics development have just been finalized. Thereby, the assembly of a first prototype laser will now start, followed by software development, optical performance tests, and integration into the microscope prototype system.
By integration of a NIR excitation laser, complementary optics, mirrors, and filters, the operation of the microscope prototype platform has been extended beyond the visible, into the NIR range. Moreover, integration of a first laser for label-free imaging and already available SPAD arrays into the platform prototypes has been accomplished.
A range of NIR fluorophore marker molecules have been investigated with respect to their blinking/switching properties, photostability and brightness, which are critical parameters for all forms of super-resolution microscopy. Several fluorophores, together with useful sample preparation and staining procedures, have been identified as appropriate for imaging with the next-generation super-resolution concept.
With the first super-resolution microscope prototype system, currently operating in the visible wavelength range, first rounds of pilot imaging have been performed on bacteria (pneumococci), showing the spatial localization patterns of a pneumococcal surface protein (PspC2) with a few nanometers resolution. PspC2, and its spatial distribution on the bacteria, play an important role in pneumococcal disease. Thus, this imaging, currently performed in the visible, is a first demonstration and makes us confident that with the prototypes in progress, we can extend this imaging into the NIR and then obtain overlaid label-free morphological and chemical images of bacteria.
We have designed prototypes of the next-generation fluorescence super-resolution microscopy platform, based on the MINFLUX concept, for operation in the near-infrared (NIR) wavelength range and offering one order of magnitude higher spatial resolution than current state-of-the-art super-resolution microscopes operating in the same wavelength range.
The development of new single photon avalanche detector (SPAD) arrays with enhanced sensitivity in the NIR are likewise making significant progress. Dedicated detection electronics have been modified, and successfully integrated into the microscope prototype platform. SPAD array chips with enhanced sensitivity have been designed for later integration.
A suitable setup for a pulsed, narrow-linewidth, multi-line laser for label-free cellular imaging has been identified. Following optical layout calculations, and tests and validation of concepts, the mechanical design and electronics development have just been finalized. Thereby, the assembly of a first prototype laser will now start, followed by software development, optical performance tests, and integration into the microscope prototype system.
By integration of a NIR excitation laser, complementary optics, mirrors, and filters, the operation of the microscope prototype platform has been extended beyond the visible, into the NIR range. Moreover, integration of a first laser for label-free imaging and already available SPAD arrays into the platform prototypes has been accomplished.
A range of NIR fluorophore marker molecules have been investigated with respect to their blinking/switching properties, photostability and brightness, which are critical parameters for all forms of super-resolution microscopy. Several fluorophores, together with useful sample preparation and staining procedures, have been identified as appropriate for imaging with the next-generation super-resolution concept.
With the first super-resolution microscope prototype system, currently operating in the visible wavelength range, first rounds of pilot imaging have been performed on bacteria (pneumococci), showing the spatial localization patterns of a pneumococcal surface protein (PspC2) with a few nanometers resolution. PspC2, and its spatial distribution on the bacteria, play an important role in pneumococcal disease. Thus, this imaging, currently performed in the visible, is a first demonstration and makes us confident that with the prototypes in progress, we can extend this imaging into the NIR and then obtain overlaid label-free morphological and chemical images of bacteria.
In this coherent and interdisciplinary project, we will prototype a next-generation super-resolution microscope system. While current state-of-the-art super-resolution microscopes offer ten times higher resolution than other advanced light microscopy techniques, this next-generation super-resolution microscopy offers an additional order of magnitude higher resolution than that. 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 will demonstrate 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 photonics-based technique. 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 also are likely to 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.
As a lead application, we will demonstrate 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 photonics-based technique. 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 also are likely to 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.