CE42 - Capteurs, instrumentation

Polarized super-resolution imaging for live conformational imaging – 3DPolariSR

Polarized super-resolution microscopy for live conformational imaging

Development of a microscopy module and associated fluorescent labels for the detection of conformational changes of proteins involved in cell adhesion by single molecule orientation and localization microscopy (SMOLM).

Development of a microscope prototype and appropriate labelling for polarized imaging of localization and orientation of single molecules

The 3DPolariSR project aims at imaging the orientational modifications of proteins in polarized super-resolution fluorescence imaging. The consortium aims to build a prototype in the form of a module adaptable to optical microscopy, eventually transferred to the industrial partner. A feasibility study will be conducted based on the know-how developed by the coordinator (partner P1) on the use of light polarization in microscopy, to measure in an unambiguous way the 3D spatial localization, the 3D average orientation and the extent of angular fluctuations of a fluorescent emitter. The evaluation of the efficiency of the estimators will be performed for two possible optical strategies (WP1). This tool will be accompanied by the development of a robust algorithm for high precision orientation and spatial imaging of single molecules (WP2). This development will allow to validate the molecular tools developed jointly by the chemist (P3) and biologist (P4) partners, to lead to generic protein labeling for which the fluorophore is bound as rigidly as possible to the protein of interest (WP3). Finally, these microscopy and molecular tools will be applied by the P4 partner to study the orientation behavior of proteins of cellular adhesion sites, whose organization is still poorly known (WP4).

The methods implemented in this project are
- Single molecule localization/orientation detection modules, by (i) polarized image split detection (4polarization) and (ii) PSF engineering detection
- Image analysis software associated to these modules
- Biological constructs of fluorescent proteins and tags attached to proteins of interest of cell adhesion sites
- Analysis of orientation and localization data on cell adhesion site proteins, under conditions of stimulation of adhesion activity

- WP1 (instrumentation P1-P2): the 'image splitting' device (4polar) has been validated in its 'home made' version (P1), and used to quantify the degree of orientation and the 3D orientation of fluorophores. A study on grafted origami of cyanine fluorophores validated the fluorophore attachment approach that can be used as reference orientations for the technique (ETH-Zurich collaboration, publication submitted). An abbelight version (P2) of the module to be adapted for the 4polar was transferred to Marseille (P1). Tests have shown the need to adapt the optics and optomechanics, in particular by replacing the mirrors with coatings that are less disruptive for polarization (protected Ag instead of dielectric) (work in progress). The «PSF engineering« device (BFP filtering) has been upgraded to be used in multi-wavelength, its validation is in progress (P1).
- WP2 (software P1-P2): A version of the 4polar image processing algorithm is currently being improved (the image analysis currently works by an estimation of intensities that can be further optimized to fit the shape of the BFP and access the axial position of the molecules, in addition to its orientation and lateral position) (P1,P2). Two approaches are being studied: deep learning (P2) and PSF fitting (P1).
- WP3 (biomolecular probes P1-P3): Different strategies are being evaluated to create a rigid link between a fluorescent protein reporter and a protein of interest. Different fluorescent reporters (HaloTag, FAST) and different rigidification strategies (use of circular permutation, creation of rigid helical linkers) are being validated. We performed a truncation screen of mEos3.2 (widely used for sptPALM (P3)), which showed that we can remove up to 4 residues on the N-terminal side and 9 residues on the C-terminal side without losing fluorescence, while keeping the photoconvertible capacity.
- WP4 (biology P1-P4): the new construct described above, based on mEos3.2, was tested by sptPALM in living cells in Bordeaux (P4) and proved to be functional in three tests: (i) localization to focal adhesions, (ii) activation by manganese and (iii) diffusive behavior measured by sptPALM. In addition, experiments performed by P4 on the abbelight module (P2) present in its premises in Bordeaux have validated the capacity of 3D localization imaging by SAF (super critical angle filtering imaging) in adhesion sites. This module will then be modified to add the functionality of a 4 polarization projection (current test at P1).

- WP1: In the next period of the project, the partners will finalize the development of the 4polarization imaging prototype (image splitter) and continue the development of the imaging by PSF engineering. The 4polarization prototype will be used for the first tests on the constrained orientation constructions will be tested
- WP2 : the image analysis codes by deep learning and PSF fitting will be finalized and implemented in a software platform associated with the polarized detection module.
- WP3 : constructs based on photoactivatable proteins and tags will be fabricated and tested for their brightness and orientational constraint.
- WP4: the first orientational sptPALM measurements will be performed on actin, talin and integrin proteins in cell adhesion sites.

1. Carla Silva Martins et al. Human septins in cells organize as octamer-based filaments mediating actin-membrane anchoring (Submitted to J Cell Bio 2022) – bioRxiv doi: doi.org/10.1101/2022.02.23.481653
2. C. Rimoli, C. Valades Cruz, V. Curcio, M. Mavrakis, S. Brasselet. 4polar-STORM polarized super-resolution imaging of actin filament organization in cells. (bioRxiv 2021.03.17.435879 (2021)) Nat. Communications 13, 301 (2022). DOI: 10.1038/s41467-022-27966-w

Following conformational changes of proteins and the way they assemble together at the nanometric scale in live cells and tissues are key elements for biology and biomedical sciences. Yet it is still today a real challenge, which requires to measure not only where proteins are localized, but also how they are oriented. 3DPolariSR proposes to tackle this problem by surpassing the state of the art in single molecule localization super-resolution imaging, using polarized fluorescence detection. The consortium will combine its expertise in optics, instrumentation, data processing, proteins engineering and biology, to build a solution for the optical microscopy market, to be transferred to its industrial partner. A feasibility study will be performed on the basis of the know-how developed by the coordinator (partner P1) on the use of light polarization in microscopy, to measure in a non-ambiguous way the spatial 3D localization, the mean 3D orientation, and the angular extent of the fluctuations of a single fluorescent emitter. The evaluation of the efficiency of estimators for several possible optical strategies (image plane polarized split, manipulation of the Fourier plane by phase plates) will permit to determine the optimal optical set-up to be transposed as a prototype by the industrial partner P2 (WP1). This tool will be accompanied by the development of a dedicated robust software for the orientational and spatial high precision imaging of single molecules (WP2). The final product, combination of technique and software, will benefit from the experience of P2 in the commercialization of modular systems for super-resolution imaging. This development will permit to validate the molecular tools jointly developed by the chemist (P3) and biologist (P4) partners, with the goal of achieving a generic way to label proteins of interest in which fluorophores are rigidly attached (WP3). At last, both microscopy and molecular tools will be applied by P4 for the study of the orientational behaviour of proteins of the cell adhesion sites, which organization is still poorly known (WP4). This novel tool will permit to establish the role of spatio-temporal conformational modifications of the proteins involved in the cell mechanotransduction. The project will, at a larger scale, lead to a new technological solution for super-resolution imaging of the conformation and organization of proteins in cells and tissues.

Project coordination

Sophie BRASSELET (Centre National de la Recherche Scientifique Délégation Provence et Corse_Institut Fresnel)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IINS INSTITUT INTERDISCIPLINAIRE DE NEUROSCIENCES
CNRS DR12_UMR7249 Centre National de la Recherche Scientifique Délégation Provence et Corse_Institut Fresnel
Abbelight AbbeLight / Direction R&D
LBM Laboratoire des biomolécules

Help of the ANR 637,076 euros
Beginning and duration of the scientific project: January 2021 - 48 Months

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