Periodic Reporting for period 3 - NANO4LIFE (High-throughput 4D imaging for nanoscale cellular studies)
Reporting period: 2023-01-01 to 2024-06-30
Understanding the structure and function of biological processes is key for comprehending life as whole and for advancing in the fields of medicine and food production. Fluorescence microscopy is an invaluable tool for such studies, as it provides high specificity and contrast for the observation of cellular components, such as lipids, DNA, RNA and proteins. These are usually tagged with fluorescent molecules in a minimally invasive fashion, even allowing the study of live specimens. The resolution of classical fluorescence microscopy is limited to the hundreds of nanometers due to the diffraction of light, whereas the molecules in question are hundreds of times smaller; however, higher resolutions were unlocked with the development of the so-called super-resolution (SR) methodologies.
These techniques rely on switching the emission state of fluorophores, such that few or only one of them emit light simultaneously, and somehow determining their exact position. Multiple variants of these techniques have emerged, achieving resolutions in the order of 10 nm to 100 nm and revealing details of subcellular organelles and new structures, as well as ultrastructural anatomy in tissue. This breakthrough transformed the observation of biological specimens, granting the Nobel Prize in Chemistry in 2014 to the developers of SR.
This project focuses on MINFLUX, a single-molecule localization strategy that uses elements of information theory to maximize the spatio-tempral performance. In MINFLUX, the location of an emitter is sequentially probed with a beam of excitation light that features a zero of intensity and its position is then ‘triangulated’. The performance of this methods reaches the single-nanometer level and, when applied to tracking, it can boost the temporal resolution by hundred-fold. The goals of this project are to advance the state-of-the-art of nanoscopy by developing a platform that can image and track biological specimens in three-dimensions with isotropic single digit nanometer resolution, in a high throughput and time resolved fashion.
These techniques rely on switching the emission state of fluorophores, such that few or only one of them emit light simultaneously, and somehow determining their exact position. Multiple variants of these techniques have emerged, achieving resolutions in the order of 10 nm to 100 nm and revealing details of subcellular organelles and new structures, as well as ultrastructural anatomy in tissue. This breakthrough transformed the observation of biological specimens, granting the Nobel Prize in Chemistry in 2014 to the developers of SR.
This project focuses on MINFLUX, a single-molecule localization strategy that uses elements of information theory to maximize the spatio-tempral performance. In MINFLUX, the location of an emitter is sequentially probed with a beam of excitation light that features a zero of intensity and its position is then ‘triangulated’. The performance of this methods reaches the single-nanometer level and, when applied to tracking, it can boost the temporal resolution by hundred-fold. The goals of this project are to advance the state-of-the-art of nanoscopy by developing a platform that can image and track biological specimens in three-dimensions with isotropic single digit nanometer resolution, in a high throughput and time resolved fashion.
The progress of the project in the first phase has been focused on establishing in the group expertise in mathematical modelling, optics, electronics, software development, fluorescent probes photophysics, and labelling strategies.
We have so far designed a high-throughput localization instrument and we have also designed, implemented and assembled a state-of-the-art MINFLUX instrument that can image and track molecules in the nanometer/subnanometer regime, in three dimensions. We devised novel scanning technologies and localization strategies, both surpassing current reported performance. These also simplify implementation, and we foresee possible adoption by instrumentation developers.
We have so far designed a high-throughput localization instrument and we have also designed, implemented and assembled a state-of-the-art MINFLUX instrument that can image and track molecules in the nanometer/subnanometer regime, in three dimensions. We devised novel scanning technologies and localization strategies, both surpassing current reported performance. These also simplify implementation, and we foresee possible adoption by instrumentation developers.
We expect to follow on these development by implementing adaptive tracking modalities and high speed imaging schemes, to foster powerful biological collaborations with the goal of studing nuclear transport, including RNA export, proteasome import and molecular conformations of distinct machinery.