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Nanometer precise vertical localization of single particles with standard fluorescent microscope in biological samples

Our aim was to establish and validate an easy‐to‐use application for standard fluorescence microscopy, which allows vertical localization of single fluorescent particles with nanometer precision. Vertical localization of single fluorescent particles can be performed with precision beyond the resolution limit by decoding the information contained in out‐of‐focus images of the particle because the size of the images is proportional to out‐of‐focus distance. Images of single fluorescent particles at defined defocus distances were used to calibrate the approach using whole diffraction patterns (WP) and outermost ring diameters (OR). Precision and validity of the WP‐ and OR‐approach were determined by in house software using independent images at defined defocus distances. In addition, Brownian motion tracking and near‐wall particle image velocymetry were performed. WP and OR yielded similar precision (6.3 nm vs. 7.4 nm) for optimal imaging conditions, underexposed images or images with overlapping patterns. WP compared to OR proved to be more robust and precise in the presence of cells and image distortions.