Final Report Summary - DR-NANO (Depth-Resolved Optical Nanoscopy)
The overall objective of the project is to develop a fluorescence microscope for sub-diffraction-limit visualization in thick samples. The microscope is based on the illumination of a confined layer in the specimen and 4pi detection of the emitted fluorescence to localize fluorescent probes in three dimensions from specific layers in the sample. The anticipated use of the microscope is in the characterization of the nanoenvironment of soft bio-materials and in biological imaging.
Since the beginning of the project, the illumination and detection paths of the microscope have been developed and optimized and depth-resolved, two-dimensional imaging and tracking of fluorescent probes in thick bio-gels and F-actin solutions have been demonstrated using a unique multiple particle tracking analysis framework developed in the project. Furthermore, the use of photoactivatable fluorescent molecules with the proposed microscope and the molecular localization algorithm developed in the project has been tested for nano-imaging in thick samples. Finally, the performance of the 4pi detection path of the microscope for axial localization has been explored experimentally and has shown a 13-nm axial localization precision, presenting a promising path towards sub-diffraction-limit imaging and tracking in thick samples in three dimensions.
The developed microscope presents a new investigational tool for the analysis of the nanoenvironment of complex material and it is expected that the techniques and knowledge gained throughout the project will promote many other studies of sub-diffraction-limit visualization deep in biological and physical systems as well as will contribute to European excellence and European competitiveness in this important field of optical nano-imaging.
Since the beginning of the project, the illumination and detection paths of the microscope have been developed and optimized and depth-resolved, two-dimensional imaging and tracking of fluorescent probes in thick bio-gels and F-actin solutions have been demonstrated using a unique multiple particle tracking analysis framework developed in the project. Furthermore, the use of photoactivatable fluorescent molecules with the proposed microscope and the molecular localization algorithm developed in the project has been tested for nano-imaging in thick samples. Finally, the performance of the 4pi detection path of the microscope for axial localization has been explored experimentally and has shown a 13-nm axial localization precision, presenting a promising path towards sub-diffraction-limit imaging and tracking in thick samples in three dimensions.
The developed microscope presents a new investigational tool for the analysis of the nanoenvironment of complex material and it is expected that the techniques and knowledge gained throughout the project will promote many other studies of sub-diffraction-limit visualization deep in biological and physical systems as well as will contribute to European excellence and European competitiveness in this important field of optical nano-imaging.