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

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posted on 2023-11-30, 20:19 authored by Martin Rieu, Thibault Vieille, Gaël Radou, Raphaël Jeanneret, Nadia Ruiz, Bertrand Ducos, Jean-François Allemand, Vincent Croquette
Super-resolutive 3D tracking, such as PSF engineering or evanescent field imaging has long been used to track microparticles and to enhance the throughput of single molecules force spectroscopic measurements. However, current methods present two drawbacks. First, they lack precision compared with optical tweezers or AFM. Second, the dependence of their signal upon the position is complex creating the need for a time-consuming calibration step. Here, we introduce a new optical technique that circumvents both issues and allows for a simple, versatile and efficient 3D tracking of diluted particles while offering a sub-nanometer frame-to-frame precision in all three spatial directions. The principle is to combine stereoscopy and interferometry, such that the z (axial) position is measured through the distance between two interferometric fringe patterns. The linearity of this stereoscopy technique alleviates the need for lookup tables while the structured interferometric pattern enhances precision. On the other hand, the extended spatial footprint of this PSF maximizes the number of photons detected per frame without the need of fancy cameras, nor the need for complex hardware. Hence, thanks to its simplicity and versatility, we believe that SDI (Stereo Darkfield Interferometry) technology has the potential to significantly enhance the spreading of 3D tracking. We demonstrate the efficiency of this technique on various single-molecule measurements thanks to magnetic tweezers. In particular we demonstrate the precise quantification of two-state dynamics involving axial steps as short as 1 nm. We then show that SDI can be directly embedded in a commercial objective providing a means to track multiple single cells in 3D .

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