New microscope opens up the nanoenvironment
The aim of the DR-NANO (Depth-resolved optical nanoscopy) project was to develop a fluorescence microscope capable of studying of soft-biomaterials and conduct biological imaging. This was achieved by creating a nanoenvironment fluorescence microscope capable of sub-diffraction-limit visualisation in thick samples. Conventional fluorescence microscopy techniques, such as confocal and two-photon microscopy, do not have the desired spatial resolution required for measurement at the nanoscale. However, techniques based on state-of-the-art 3D super-resolution optical microscopy allow limited penetration to a depth of less than 5 microns. Researchers therefore developed a new microscope based on the illumination of a confined layer in a specimen and 4Pi detection of the emitted fluorescence. This enabled the localisation of fluorescent probes in three dimensions from specific layers in the sample. The illumination and detection in the microscope was developed, tested and depth-resolved. The two-dimensional imaging and tracking of fluorescent probes was demonstrated in thick biogels and F-actin solutions using a multiple particle tracking framework unique to the project. The new microscope was also used to test the use of photoactivatable fluorescent molecules for nanoimaging in thick samples. This was done in combination with the molecular localisation algorithm developed by the project. Finally, scientists examined the performance of the microscope’s 4Pi protection path for axial localisation, which showed 13-nm axial localisation precision. This proved promising for sub-diffraction-limit imaging and tracking in thick samples in 3 dimensions. The microscope developed by the project team represented a new investigative tool for the detailed study of the nanoenvironment of complex materials. It will open up new possibilities for the precise measurement of the diverse structure and mechanical properties of soft matter at extended depths. DR-NANO will ultimately assist in the development of superior biomaterials and nanomedical therapies, thereby contributing to European excellence and competitiveness in the important field of optical nanoimaging.
Keywords
Fluorescence, microscope, DR-NANO, soft-biomaterials, sub-diffraction-limit visualization, fluorescent probes, nanoimaging, 4Pi, nanomedical therapies