Single Molecule 3D Orientation and Localization Microscopy (SMOLM) via ratiometric 4-polarization projection microscopy on dense actin structures

Session

Session 5 - Super-resolution and Nanoscale Imaging

Authors

Charitra Sree Senthil Kumar (1), Miguel Sison (1), Cesar Augusto Valades-Cruz (1, 2), Luis Arturo Aleman-Castaneda (1), Manos Mavrakis (1), Sophie Brasselet (1)

Affiliations

1. Aix Marseille Univ, CNRS, Centrale Med, Institut Fresnel, Marseille
2. Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan

Keywords

1. super-resolution micrscopy
2. polarization microscopy
3. single molecule orientation and localization microscopy SMOLM
4. actin
5. STORM

Abstract text

The dynamics of intracellular processes rely heavily on the precise organization of biomolecules involved and their orientation relative to one another. Single-molecule localization microscopy (SMLM) has been pivotal in advancing the study of these processes, providing imaging resolutions down to the single-molecule level. However, a more thorough comprehension of these processes necessitates not only knowledge of molecular positions, but also their orientations, whose determination is the goal of Single Molecule Orientation and Localization Microscopy (SMOLM). These parameters being inherently linked within the microscope’s point spread function (PSF), various techniques based on PSF engineering and PSF fitting have been developed to retrieve both the orientation and position of the emission dipole of fluorescent molecules [1]. Nonetheless, many of these approaches entail intricate implementation and time consuming data processing, which are delicate to apply to dense cell samples.

Recently, we introduced a polarization-splitting technique that enables the retrieval of the 2D orientation and wobbling extent of fluorescent molecules using image splitting and ratiometric intensity estimations onto four polarization channels, with minimal PSF deformation [2]. Here, we extend the capabilities of this approach to full 3D orientation determination, based on a straightforward back focal plane filtering, exploiting the fact that the radiation distribution of the emission dipole is dependent on the molecules' off-plane tilt angle [3]. The performance of the method is demonstrated in model systems based on DNA origamis [4] and lipid bilayers. We further demonstrate actin orientation imaging in dense structures such as tumor cells’ lamellipodia and actin podosome 3D structures in macrophages [5]. Using light microscopy, we evidence actin off-plane tilt orientations in these structures, which was so far only accessible through electron microscopy [5]. Additionally, we investigate the impact of different data processing methods on the accuracy and precision of the measured orientation parameters [5].

References

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orientation and localization microscopy. Minireview Optica 10 (11), 1486-1510 (2023).
2. Rimoli, C.V. et al. 4polar-STORM polarized super-resolution imaging of actin filament organization in cells. Nat
Commun 13, 301 (2022).
3. Hohlbein, J. and Hübner, C.G., Three-dimensional orientation determination of the emission dipoles of single
molecules: The shot-noise limit. J. Chem. Phys. 129, 094703 (2008).
4. Adamczyk, A.K., et al. DNA self-assembly of single molecules with deterministic position and orientation. ACS Nano
16 (10), 16924-16931 (2022).
5. Charitra S. Senthil Kumar, M. Sison et al. in prep. Single molecule 3D orientation and localization imaging using
simple ratiometric polarization splitting.