Periodic Reporting for period 3 - BrightEyes (Multi-Parameter Live-Cell Observation of Biomolecular Processes with Single-Photon Detector Array)
Berichtszeitraum: 2022-09-01 bis 2024-02-29
Understanding how biomolecules behave is the holy grail of cell biology research. The cell is a crowded and ever-changing environment where biomolecules jostle around, interact, concentrate, change the structure, and organize in a hierarchical way to carry out all the processes that regulate life. Deciphering the bio-molecular processes underlying a cell's physiology is fundamental to understanding human health, ageing, and disease. However, observing such processes is far from simple: Biomolecular processes occur over different temporal and spatial scales, are heterogeneous in time and space, involve the cooperation of several biomolecules and sub-cellular components, and take place in only partially transparent cells. This complexity makes the observation of biomolecular processes in a living cell one of the biggest challenges for "the microscope makers".
Single-molecule (SM) microscopy techniques have the unique ability to observe the behaviour of an individual biomolecule at a time – which eliminates all the implicit spatial and temporal averages of ensemble methods. Specifically, by recording the motion of a single fluorophore-attached biomolecule, SM tracking reveals the bio-molecular dynamics and the interactions underlying a process of interest.
However, any state-of-the-art SM tracking technique is a trade-off between spatiotemporal resolution and range, information content, working conditions and throughput.
The BrightEyes project aims to leverage novel single-photon array detectors to develop a universal, large-information content, high-resolution SM approach for living (multi-) cellular environments. This approach will correlate the biomolecule's motion with other fluorescence spectroscopy parameters, which allow monitoring of the biomolecule's chemical nano-environment, structural changes, and relations with other biomolecule species. Furthermore, the simultaneous imaging of the micro-environment correlates with how the tracked biomolecule fits into the sub-cellular structures.
This unique technology will reveal, with unprecedented details, how individual molecules interact, self-organize, and change in structure to carry out the fundamental processes of life. For example, this technology will help understand the cause of the amyotrophic lateral sclerosis (ALS) disease and how RNA-based therapeutics can help find a cure.
Single-molecule (SM) microscopy techniques have the unique ability to observe the behaviour of an individual biomolecule at a time – which eliminates all the implicit spatial and temporal averages of ensemble methods. Specifically, by recording the motion of a single fluorophore-attached biomolecule, SM tracking reveals the bio-molecular dynamics and the interactions underlying a process of interest.
However, any state-of-the-art SM tracking technique is a trade-off between spatiotemporal resolution and range, information content, working conditions and throughput.
The BrightEyes project aims to leverage novel single-photon array detectors to develop a universal, large-information content, high-resolution SM approach for living (multi-) cellular environments. This approach will correlate the biomolecule's motion with other fluorescence spectroscopy parameters, which allow monitoring of the biomolecule's chemical nano-environment, structural changes, and relations with other biomolecule species. Furthermore, the simultaneous imaging of the micro-environment correlates with how the tracked biomolecule fits into the sub-cellular structures.
This unique technology will reveal, with unprecedented details, how individual molecules interact, self-organize, and change in structure to carry out the fundamental processes of life. For example, this technology will help understand the cause of the amyotrophic lateral sclerosis (ALS) disease and how RNA-based therapeutics can help find a cure.
A key element for the success of the whole BrightEyes project is the realisation of the microscope photo-detection unit. We dedicated a part of the first-period project to realising a small (i.e. 7x7 pixel) single-photon camera (i.e. detector array) able to record fluorescence light photon-by-photon and with a temporal precision of a hundred picoseconds.
We successfully used the novel camera to implement all three optical methods composing the BrightEyes optical architecture: single-molecule tracking, fluorescence correlation spectroscopy, and sub-cellular imaging. Thanks to the camera's high temporal resolution, we correlate all three methods with the fluorescence lifetime assay, thus always providing information about the biomolecule's structure, interactions and chemical nano-environment.
Having only a few elements, our camera images only a small bi-dimensional field-of-view within the specimen. In the context of single-molecule tracking, we extended the field-of-view by using the three-dimensional beam scanning apparatus of the microscope to implement a real-time feedback system: The microscope probing region follows the biomolecule as a sweet spot follows an actor on the stage.
If the bio-molecule moves too fast, the scanning apparatus cannot follow it. Thus, we used the new camera to implement fluorescence fluctuation spectroscopy. The microscope's probing region is fixed, and the fluorescence light fluctuations induced by the many bio-molecules entering and exiting this region are analysed to infer their diffusion coefficient. The image of the probing region allows for further information about the bio-molecule dynamics, such as its diffusion modality.
Single-molecule tracking and fluorescence-fluctuation spectroscopy provide complementary dynamics information. The former reveals spatiotemporal molecule heterogeneities but cannot follow fast processes. The second reveals dynamics faster than any other optical technique but provides only average behaviours. On the other side, following the changes of the large sub-cellular structures around the investigated molecules does not need high temporal resolution but high spatial resolution. Laser-scanning microscopy satisfies this temporal need. We show that our camera allows implementing image scanning microscopy and its combination with STED microscopy, thus improving the resolution of laser-scanning microscopy while maintaining live-cell compatibility.
We successfully used the novel camera to implement all three optical methods composing the BrightEyes optical architecture: single-molecule tracking, fluorescence correlation spectroscopy, and sub-cellular imaging. Thanks to the camera's high temporal resolution, we correlate all three methods with the fluorescence lifetime assay, thus always providing information about the biomolecule's structure, interactions and chemical nano-environment.
Having only a few elements, our camera images only a small bi-dimensional field-of-view within the specimen. In the context of single-molecule tracking, we extended the field-of-view by using the three-dimensional beam scanning apparatus of the microscope to implement a real-time feedback system: The microscope probing region follows the biomolecule as a sweet spot follows an actor on the stage.
If the bio-molecule moves too fast, the scanning apparatus cannot follow it. Thus, we used the new camera to implement fluorescence fluctuation spectroscopy. The microscope's probing region is fixed, and the fluorescence light fluctuations induced by the many bio-molecules entering and exiting this region are analysed to infer their diffusion coefficient. The image of the probing region allows for further information about the bio-molecule dynamics, such as its diffusion modality.
Single-molecule tracking and fluorescence-fluctuation spectroscopy provide complementary dynamics information. The former reveals spatiotemporal molecule heterogeneities but cannot follow fast processes. The second reveals dynamics faster than any other optical technique but provides only average behaviours. On the other side, following the changes of the large sub-cellular structures around the investigated molecules does not need high temporal resolution but high spatial resolution. Laser-scanning microscopy satisfies this temporal need. We show that our camera allows implementing image scanning microscopy and its combination with STED microscopy, thus improving the resolution of laser-scanning microscopy while maintaining live-cell compatibility.
All methods developed within the BrightEyes project are well beyond the state-of-the-art in their respective contexts, i.e. single-molecule tracking, fluorescence correlation spectroscopy, and raster scanning imaging.
The single-photon camera introduced by the BrightEyes project has triggered a new paradigm: single-photon microscopy. Because laser-scanning microscopy builds the image point-by-point, it naturally multiplexes the information in time, reducing the photon-flux that the data acquisition needs to sustain. This condition opens to an efficient photon-by-photon recording with a series of temporal and spatial tags -- to which only our camera provides access. Access to this information allows to improve the performance of many established techniques and introduce brand-new ones. We have shown how to reduce the dose of light requested by stimulated emission depletion microscopy. We have introduced fluorescence lifetime fluctuation spectroscopy: For the first time, we have correlated the bio-molecules dynamics model with the fluorescence lifetime. Together with the vast interest of many microscopy manufacturers, these results reinforce our perspective on seeing our single-photon camera in any laser-scanning microscope.
At the project's current stage, we implemented the three classes of methods on three different microscopes. However, we are confident that we will implement all methods on the same system, thus opening to experiments that can provide a complete picture of the bio-molecular processes of interest.
An essential aspect of the project's current state is that we have already started using the developed technology in relevant applications. In the context of the Amyotrophic Lateral Sclerosis (ALS) neurodegenerative disease, we are investigating the dynamics formation of protein aggregates (i.e. stress granules) in motor neurons carrying ALS-associated FUS gene mutations. By correlating the dynamics of a protein involved in the construction of the stress granules with its fluorescence lifetime -- to indicate protein-protein interaction, we can reveal the early stages of stress granules and shine new light on this process.
At the end of the project, we expect to use the BrightEyes system to provide a complete picture of this phenomenon. For example, we will integrate the single-molecule tracking method to investigate the aggregating proteins' behaviour with other molecules, such as circular RNAs.
The single-photon camera introduced by the BrightEyes project has triggered a new paradigm: single-photon microscopy. Because laser-scanning microscopy builds the image point-by-point, it naturally multiplexes the information in time, reducing the photon-flux that the data acquisition needs to sustain. This condition opens to an efficient photon-by-photon recording with a series of temporal and spatial tags -- to which only our camera provides access. Access to this information allows to improve the performance of many established techniques and introduce brand-new ones. We have shown how to reduce the dose of light requested by stimulated emission depletion microscopy. We have introduced fluorescence lifetime fluctuation spectroscopy: For the first time, we have correlated the bio-molecules dynamics model with the fluorescence lifetime. Together with the vast interest of many microscopy manufacturers, these results reinforce our perspective on seeing our single-photon camera in any laser-scanning microscope.
At the project's current stage, we implemented the three classes of methods on three different microscopes. However, we are confident that we will implement all methods on the same system, thus opening to experiments that can provide a complete picture of the bio-molecular processes of interest.
An essential aspect of the project's current state is that we have already started using the developed technology in relevant applications. In the context of the Amyotrophic Lateral Sclerosis (ALS) neurodegenerative disease, we are investigating the dynamics formation of protein aggregates (i.e. stress granules) in motor neurons carrying ALS-associated FUS gene mutations. By correlating the dynamics of a protein involved in the construction of the stress granules with its fluorescence lifetime -- to indicate protein-protein interaction, we can reveal the early stages of stress granules and shine new light on this process.
At the end of the project, we expect to use the BrightEyes system to provide a complete picture of this phenomenon. For example, we will integrate the single-molecule tracking method to investigate the aggregating proteins' behaviour with other molecules, such as circular RNAs.