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Super-resolutive 3D tracking, such as PSF engineering or evanescent fieldimaging has long been used to track microparticles and to enhance thethroughput of single molecules force spectroscopic measurements. However,current methods present two drawbacks. First, they lack precision compared withoptical tweezers or AFM. Second, the dependence of their signal upon theposition is complex creating the need for a time-consuming calibration step.  Here, we introduce a new optical technique that circumvents both issues andallows for a simple, versatile and efficient 3D tracking of diluted particleswhile offering a sub-nanometer frame-to-frame precision in all three spatialdirections. The principle is to combine stereoscopy and interferometry, suchthat the z (axial) position is measured through the distance between twointerferometric fringe patterns. The linearity of this stereoscopy techniquealleviates the need for lookup tables while the structured interferometricpattern enhances precision. On the other hand, the extended spatial footprintof this PSF maximizes the number of photons detected per frame without the needof fancy cameras, nor the need for complex hardware. Hence, thanks to itssimplicity and versatility, we believe that SDI (Stereo DarkfieldInterferometry) technology has the potential to significantly enhance thespreading of 3D tracking.  We demonstrate the efficiency of this technique on various single-moleculemeasurements thanks to magnetic tweezers. In particular we demonstrate theprecise quantification of two-state dynamics involving axial steps as short as1 nm. We then show that SDI can be directly embedded in a commercial objectiveproviding a means to track multiple single cells in 3D .

science advances

Stereo Darkfield Interferometry : a versatile localization method for  subnanometer force spectroscopy of single molecules and 3D-tracking of single  cells