Nanolocalization measurements in spatially modulated illumination microscopy using two coherent illumination beams

The determination of the 3D nanostructure of specific chromatic regions is highly relevant for an improved understanding of the functional topology of the genome. The use of different spectral signatures for the labeling and high accuracy nanodistance measurements in the spectral precision distance microscopy mode allows the investigation of the topology of such targets in 3D conserved nuclei. To obtain the required high-accuracy nanolocalization of small targets, interferometric illumination is a well established and reliable tool. New approaches use spatially modulated illumination (SMI) in various ways. In our laboratory a stage controlled optical sectioning through the object was applied. In this case the SMI point spread function is the product of the axial illumination modulation and of the conventional PSF of the microscope objective. Using this approach and an appropriate analysis algorithm, the position of 'point-like' fluorescent objects was determined with an axial localization precision in the range of 2nm. To provide a reliable high precision localization performance also for long time measurements, thermally invariant mounting devices have been developed for the SMI-system. Using this improved system, it was possible to measure the thermal shift induced by the microscope objective itself.