Simultaneous 3D localization and 2D orientation determination for single-molecules super resolution microscopy

Super-resolution (SR) microscopy have revolutionized our understanding of the biological processes at the molecular level. Imaging single molecules and localizing their center with high precision allows the reconstruction of pointillist image of the sample with a resolution beyond the diffraction limit¹²³ [1,2,3]. In order to capture the 3D nanoscale morphology of the whole cell, multifocus microscopy (MFM) has been proposed to instantaneously acquire the 3D localization of single molecules (SM) in cells within a volume of a few micrometers⁴⁵ [4,5]. Still additional information such as molecular orientation can provide supplementary information relating the local molecular organization and arrangement to the biological function. To retrieve the orientation of SM, two main strategies have been developed: PSF-engineering based methods and polarimetry measurements⁶ [6]. In the second strategy, a polarization-splitting scheme combined with SMLM allows to determine both the 2D localization and orientation of SM.

The aim of this internship is to retrieve the orientation measurement simultaneously with 3D positional information. The method will rely on combining MFM with polarization measurements at the different focal plane.

The goal of the internship is to implement this technique and to test it on real biological samples.

Tasks:

• Optimization of the optical setup
• Work on the reconstruction and analysis algorithms
• Perform experiments on reference test samples as well as biological samples

Students with backgrounds in physics, optics or math can apply. Good motivation and desire to learn are important.

1. Betzig, E., et al., Imaging intracellular fluorescent proteins at nanometer resolution. Science (2006). ↩︎
2. Rust, M.J., M. Bates, and X. Zhuang, Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM). Nat. Methods (2006). ↩︎
3. Schnitzbauer, J., Strauss, M., Schlichthaerle, T. et al., Super-resolution microscopy with DNA-PAINT. Nat. Protoc. (2017). ↩︎
4. Hajj, B. et al., Whole-cell, multicolor superresolution imaging using volumetric multifocus microscopy. Proc. Natl. Acad Sci. USA 111 (2014). 10.1073/pnas.1412396111. ↩︎
5. Hajj, B. et al. Highly efficient multicolor multifocus microscopy by optimal design of diffraction binary gratings. Sci. Rep. 7 (2017). 10.1038/s41598-017-05531-6. ↩︎
6. Rimoli, C. et al. 4polar-STORM polarized super-resolution imaging of actin filament organization in cells. Nat. Commun. 13 (2022). 10.1038/s41467-022-27966-w. ↩︎