Optical imaging techniques and probe development have boosted knowledge on genomics and proteomics. High quality imaging techniques and novel sources of contrast are being developed, however 3D information and more importantly quantification is still lacking especially at a resolution higher than that provided by confocal microscopy. It is now high time to develop more elaborate approaches that will improve resolution, sensitivity and specificity of real-time in-vivo imaging of gene expression and biological processes in 3D.
We propose to develop a novel microscopy technique based on tomographic approaches for 3D in-vivo imaging of biological processes. Optical tomography provides isotropic optical resolution in all three dimensions of space; this resolution is however limited by diffraction to about 250nm for visible light. To overcome this impasse, structured illumination will be implemented, thus improving the resolution drastically compared to conventional microscopy techniques. By making use of saturate d patterned fluorescence excitation, the resolution barrier can be overcome altogether.
This technique will be applied to imaging structure and dynamics at the cell nuclear level. Subsequently its potential has to be tested in the analysis of complex systems such as the drosophila melanogaster. To that end, a custom-made microscope will be extended for structured and saturated fluorescence excitation, and corresponding inverse mapping algorithms will be developed to reconstruct the objects or the fluorescence dye distribution respectively from the 3D images. This new optical imaging device will then be used to quantitatively study nuclear and sub-cellular structure, organisation and dynamics in 3D.
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