Final Report Summary - SMARTS (Super-resolution Fluorescence Microscopy based on Artificial Mesoscopic Structures)
The overall objective of the funded research project SMArtS was to study the design and fabrication of a class of artificial materials and devices whose structural features give rise to fundamentally new optical properties and unprecedented performance.
In summary, the project achieved the initial objectives, in particular the design and fabrication of novel artificial mesoscopic structure. The optimization, fabrication, characterization and testing of biocompatibility are accomplished and published. Taking into account the transfer of knowledge activities, the milestone and deliverable produced, most of the objectives of the project are achieved and partly published. Not yet fully achieved is the final integration of the techniques in terms of an imaging platform that is complete and easy to use. However, this will be realized within the next two years after all characterizations and calibrations and optimizations are done.
In the recent years, multidisciplinary approaches bridging life and material sciences seemed to hear the wake-up call and became top priority in cross-disciplinary research. Looking at the large amount of good ideas, theoretical studies, proof of concept, experimental methods, and commercialization gives us a foretaste of what will come in near future. The importance of material sciences for life sciences applications cannot be underestimated.
Our project involved designing and nanofabricating biocompatible artificial mesoscopic structures and nanostructures that can serve as modified microscope slides for studying live cell processes. Within the framework of the project, we have recently demonstrated that fast fluorescence imaging with axial nanometer localization precision is possible using such structures. The design is relatively simple so that nanofabrication of such structures is feasible, straight forward and cost effective. Culturing cells on these modified microscope slides turned out to be unhampered following common protocols and finally allowed for an effective axial resolution of typically 10 nm within the evanescent field above the substrate in a live cell experiment. Furthermore, by exploiting the position dependent emission spectrum of fluorophores above our designed structure our approach overcomes the need for scanning. The relevant paper has been published in Proceedings of the National Academy of Sciences (Elsayad et al. 2013) with the applicant Piau Siong Tan as a co-author and Dr. Heinze as one corresponding author.
To push further, we are currently revisiting Förster resonance energy transfer (FRET) as a fascinating tool that can be performed in a Fluorescence Lifetime Imaging Microscope (FLIM) to study e.g. inter- and intramolecular dynamics of proteins in live cells. FRET is based on nonradiative dipole-dipole coupling between two marker fluorophores (called ‘donor’ and ‘acceptor’). The efficiency of this energy transfer is known to be inversely proportional to the sixth power of the distance between donor and acceptor molecule. This makes FRET an excellent sensor of small distances, however useless when larger distances need to be probed. As the resolution limit of diffraction limited fluorescence imaging techniques is around half a micrometer, there is an obvious gap between largest distance that can be probed by FRET and the smallest detail that can be visualized in an image.
An accurate way to understand the interaction between quantum emitter (QE) and electromagnetic (EM) field requires a quantum electrodynamics (QED) formalism in which both the emitter’s energy levels and the EM field are quantized. To achieve high efficient light-matter interaction in cavity based systems where large modal volumes are easily compensated by extremely narrow resonances we develop the theoretical formalism that allows us to deal with the coupling of QE to the EM modes supported by the biocompatible nanostructure. We found that that the biocompatible nanostructure is capable of supporting different surface plasmon modes, which can be used to enhance FRET efficiency (unpublished results). We demonstrated experimentally that our nanostructures can be used to amplify very low FRET signal and this also implies that the energy could transfer to larger distance between donor and acceptor (unpublished work).
In an interdisciplinary approach, this novel nanostructure-based FRET technique with enhanced operating range and efficiency will bring improved signal to noise (S/N) ratio of the final data sets. The latter is particularly important for single molecule studies where the S/N ratio is limiting, while the extended dynamic range becomes important when probing large biomolecules such as membrane receptors and their dynamics and interactions in real-time. Although previously described FRET ‘booster’ methodologies showed a strong enhancement of FRET efficiency and Förster radius, most of them are not feasible for biological applications. In contrast, our approach is fully biocompatible and can be potentially used to quantify the dynamic characteristic of signaling pathways and molecular interactions on biological (and other) membranes. Here, we have established a solid collaboration between the laboratory of Martin Lohse, who studies G-protein coupled receptors and their interactions partners via FRET and other related single molecule techniques. This group kindly provides all the constructs for live cell fluorescence microscopy, and is highly committed to this collaboration.
Of course, the goals of our current and future research plans are linked with the research activity of both the host institute and collaborators and will rise to newly developed microscope platforms that fuses new concepts such as those developed within the framework of this project with existing imaging techniques. This integration will not only help to learn more about molecular and cellular dynamics, but also open the door for new research fields and nano-technological perspectives for the host organization and will further enhance the multi-disciplinary scientific excellence of the region.
In summary, the project achieved the initial objectives, in particular the design and fabrication of novel artificial mesoscopic structure. The optimization, fabrication, characterization and testing of biocompatibility are accomplished and published. Taking into account the transfer of knowledge activities, the milestone and deliverable produced, most of the objectives of the project are achieved and partly published. Not yet fully achieved is the final integration of the techniques in terms of an imaging platform that is complete and easy to use. However, this will be realized within the next two years after all characterizations and calibrations and optimizations are done.
In the recent years, multidisciplinary approaches bridging life and material sciences seemed to hear the wake-up call and became top priority in cross-disciplinary research. Looking at the large amount of good ideas, theoretical studies, proof of concept, experimental methods, and commercialization gives us a foretaste of what will come in near future. The importance of material sciences for life sciences applications cannot be underestimated.
Our project involved designing and nanofabricating biocompatible artificial mesoscopic structures and nanostructures that can serve as modified microscope slides for studying live cell processes. Within the framework of the project, we have recently demonstrated that fast fluorescence imaging with axial nanometer localization precision is possible using such structures. The design is relatively simple so that nanofabrication of such structures is feasible, straight forward and cost effective. Culturing cells on these modified microscope slides turned out to be unhampered following common protocols and finally allowed for an effective axial resolution of typically 10 nm within the evanescent field above the substrate in a live cell experiment. Furthermore, by exploiting the position dependent emission spectrum of fluorophores above our designed structure our approach overcomes the need for scanning. The relevant paper has been published in Proceedings of the National Academy of Sciences (Elsayad et al. 2013) with the applicant Piau Siong Tan as a co-author and Dr. Heinze as one corresponding author.
To push further, we are currently revisiting Förster resonance energy transfer (FRET) as a fascinating tool that can be performed in a Fluorescence Lifetime Imaging Microscope (FLIM) to study e.g. inter- and intramolecular dynamics of proteins in live cells. FRET is based on nonradiative dipole-dipole coupling between two marker fluorophores (called ‘donor’ and ‘acceptor’). The efficiency of this energy transfer is known to be inversely proportional to the sixth power of the distance between donor and acceptor molecule. This makes FRET an excellent sensor of small distances, however useless when larger distances need to be probed. As the resolution limit of diffraction limited fluorescence imaging techniques is around half a micrometer, there is an obvious gap between largest distance that can be probed by FRET and the smallest detail that can be visualized in an image.
An accurate way to understand the interaction between quantum emitter (QE) and electromagnetic (EM) field requires a quantum electrodynamics (QED) formalism in which both the emitter’s energy levels and the EM field are quantized. To achieve high efficient light-matter interaction in cavity based systems where large modal volumes are easily compensated by extremely narrow resonances we develop the theoretical formalism that allows us to deal with the coupling of QE to the EM modes supported by the biocompatible nanostructure. We found that that the biocompatible nanostructure is capable of supporting different surface plasmon modes, which can be used to enhance FRET efficiency (unpublished results). We demonstrated experimentally that our nanostructures can be used to amplify very low FRET signal and this also implies that the energy could transfer to larger distance between donor and acceptor (unpublished work).
In an interdisciplinary approach, this novel nanostructure-based FRET technique with enhanced operating range and efficiency will bring improved signal to noise (S/N) ratio of the final data sets. The latter is particularly important for single molecule studies where the S/N ratio is limiting, while the extended dynamic range becomes important when probing large biomolecules such as membrane receptors and their dynamics and interactions in real-time. Although previously described FRET ‘booster’ methodologies showed a strong enhancement of FRET efficiency and Förster radius, most of them are not feasible for biological applications. In contrast, our approach is fully biocompatible and can be potentially used to quantify the dynamic characteristic of signaling pathways and molecular interactions on biological (and other) membranes. Here, we have established a solid collaboration between the laboratory of Martin Lohse, who studies G-protein coupled receptors and their interactions partners via FRET and other related single molecule techniques. This group kindly provides all the constructs for live cell fluorescence microscopy, and is highly committed to this collaboration.
Of course, the goals of our current and future research plans are linked with the research activity of both the host institute and collaborators and will rise to newly developed microscope platforms that fuses new concepts such as those developed within the framework of this project with existing imaging techniques. This integration will not only help to learn more about molecular and cellular dynamics, but also open the door for new research fields and nano-technological perspectives for the host organization and will further enhance the multi-disciplinary scientific excellence of the region.