Polarized fluorescence microscopy
Close collaborators : M. Mavrakis (I. Fresnel Marseille), S. Mailfert and J.J. Honorine (I. Fresnel Marseille), C. Chandre (I2M Marseille)
Light polarization can be exploited to probe the orientation of fluorescent molecules. By tuning the polarization of excitation light, we quantify the orientational order of fluorophores attached to biological proteins or to lipids within the excitation volume of a microscope objective. We have developed polarization-sensitive approaches compatible with a range of imaging modalities, including confocal microscopy, spinning-disk imaging, and two- and three-photon excitation, enabling the measurement of molecular organization at the nanoscale, in diverse biological systems. Together with this effort in instrumentation, we have developed a dedicated polarimetry-analysis open source software https://github.com/cchandre/Polarimetry. We are also pursuing the development of dedicated molecular probes specifically tailored to enable polarization-sensitive imaging in live cells and tissues.
Highlights:
- Live polarimetry imaging of actin in cells and tissues
C. Silva Martins, F. Iv, S. Kumar Suman, T. C. Panagiotou, C. Sidor, M. Ruso-López, C. N. Plancke, S. Omi, M. Gomes, A. Llewellyn, S. Reddy Bandi, L. Ramond, F. Arbizzani, C. Vaz Rimoli, F. Schnorrer, F. Robin, A. Wilde, L. LeGoff, J.-D. Pedelacq, S. Cabantous, S.A. Rincon, C. Chandre, S. Brasselet, M. Mavrakis
Genetically encoded reporters of actin filament organization in living cells and tissues
Cell 188 (9), 2540 - 2559.e27 (2025)
https://doi.org/10.1016/j.cell.2025.03.003 - Molecular order imaging in actin in the cytokinesic ring of cells during cellularization in the drosophila melanogaster embryo.
M. Mavrakis, Y. Azou-Gros, F-C. Tsai, J. Alvarado, A. Bertin, F. Iv, A. Kress, S. Brasselet, G.H. Koenderink and T. Lecuit.
Septins promote F-actin ring formation by cross-linking actin filaments into curved bundles
Nature Cell Biology 16, 322–334 (2014) - Molecular order imaging in actin in the cytokinesic ring of cells during cellularization in the drosophila melanogaster embryo.
A. Kress, X. Wang, H. Ranchon, J. Savatier, H. Rigneault, P. Ferrand, S. Brasselet
Mapping the local organization of cell membranes using generalized polarization resolved confocal fluorescence microscopy
Biophys. J. 105, 127-136 (2013)
Polarized super-resolution imaging
Close collaborators : M. Mavrakis (I. Fresnel Marseille), M. Alonso (I. Fresnel, Univ. Rochester USA), L. Aleman Castaneda (I. Fresnel)
Light polarization can be exploited to probe the orientation of fluorescent molecules. By tuning the polarization of excitation light, we quantify the orientational order of fluorophores attached to biological proteins or to lipids within the excitation volume of a microscope objective. We have developed polarization-sensitive approaches compatible with a range of imaging modalities, including confocal microscopy, spinning-disk imaging, and two- and three-photon excitation, enabling the measurement of molecular organization at the nanoscale, in diverse biological systems. Together with this effort in instrumentation, we have developed a dedicated polarimetry-analysis open source software https://github.com/cchandre/Polarimetry. We are also pursuing the development of dedicated molecular probes specifically tailored to enable polarization-sensitive imaging in live cells and tissues.
Highlights:
- Single Molecule Orientation and Localization Microscopy (SMOLM) based on polarized splitting
C. S Senthil Kumar, C. A Valades Cruz, M. Sison, A. G Vesga, J. Rey-Barroso, V. Curcio, L. A Alemán-Castañeda, M. A Alonso, R. Poincloux, M. Mavrakis, S. Brasselet
4polar3D single molecule imaging of 3D orientation in dense actin networks using ratiometric polarization splitting
Nature Communications 17, 4246 (2026)C. Rimoli, C. Valades Cruz, V. Curcio, M. Mavrakis, S. Brasselet
4polar-STORM polarized super-resolution imaging of actin filament organization in cells
Nat. Communications 13, 301 (2022) - SMOLM based on Point Spread Function (PSF) Engineering
V. Curcio, L. A. Aleman-Castaneda, T. G. Brown, S. Brasselet, M. A. Alonso
Birefringent Fourier filtering for single molecule Coordinate and Height super-resolution Imaging with Dithering and Orientation (CHIDO)
Nat. Communications 11, 5307 (2020) - Imaging of fluorophores orientation and fluctuations in filaments of the cell cytoskeleton
C.A. Valades Cruz, H. A. Shaban, A. Kress, N. Bertaux, S. Monneret, M. Mavrakis, J. Savatier, S. Brasselet
Quantitative nanoscale imaging of orientational order in biological filaments by polarized super-resolution microscopy
Proc. Natl. Acad. Sci. 113, (7) E820-E828 (2016)
Nanophotonics
Polarized optical microscopy provides a unique way to probe and control the vectorial properties of single nano-emitters, of interest for nanophotonics. Using circular polarized emission captured by a high numerical aperture objective, we have shown the possibility to use metallic nanoparticles as probes of local polarizations oriented in 3D, opening a new route for 3D nano-polarimetry. Using polarized second harmonic generation microscopy, we have performed structural analysis of metal nano-structures supporting local enhancement of optical responses, as well as inorganic nanocrystals. This properties takes advantage of the polarization dependence of second harmonic generation to nanoparticules surface and symmetry, to reveal sub-diffraction scale features related to their shape or crystalline phases.
Highlights:
- Nanoscale 3D polarimetry using plasmonic nanoparticles
I. Herrera, M. Alonso, S. Brasselet
3D Stokes polarimetric imaging at nanoscales
Nature Photonics (accepted, 2026) - arXiv.2511.11222 (2025)Herrera-Hernandez, L. Aleman-Castaneda, S. Brasselet, M. Alonso
Stokes-based analysis for the estimation of 3D dipolar emission
J. Opt. Soc. Am. B 41(11), pp. 2134-2148 (2024) - Vectorial imaging of nonlinear fields enhancements in plasmonics nanoparticles, at the nanometric scale
N.K. Balla, C. Rendon-Barraza, L.M. Hoang, P. Karpinski, E. Bermudez, S. Brasselet
Polarized nonlinear nanoscopy of metal nanostructures
ACS Photonics accepted (2017), 10.1021/acsphotonics.6b00635
Nonlinear microscopy
Due to the nonlinear vectorial coupling between incident light polarizations and molecular bonds / molecular induced dipoles directions, tunable incident polarizations lead to a modulation of nonlinear label-free signals (Second Harmonic Generation : SHG, Coherent Anti Stokes Raman Scattering : CARS, Stimulated Raman Scattering : SRS) that can be directly related to the orientational order of biological molecules, without the need of a fluorescent label. We have applied polarized nonlinear microscopy to quantitatively retrieve organizational order in collagen in tissues (by polarized SHG) and lipid structures (by polarized CARS sensitive to CH2 stretching vibrations). This methodology is highly sensitive to lipid phases but also to sub-diffraction scale morphological changes in cell membranes. Polarization resolved CARS (pCARS) is now used to image the effect of neurodegenerative diseases on fine myelin structural changes, in mice spinal cord tissues.
Highlights:
- Molecular order imaging of lipids in the myelin sheath of the mouse spinal cord axons using label free, chemically sensitive coherent anti Stokes Raman scattering
P. Gasecka, A. Jaouen, F.-Z. Bioud, H. Barbosa de Aguiar, J. Duboisset, P. Ferrand, H. Rigneault, N. Balla, F. Debarbieux, S. Brasselet
Degradation of molecular organization of myelin lipids in autoimmune demyelination probed by polarization resolved nonlinear vibrational microscopy
Biophys. J. accepted (2017) -
M. Hofer, N.K. Balla, S. Brasselet
High speed polarization resolved Coherent Raman Scattering imaging
Optica 4 (7), pp. 795-801 (2017)
Imaging in scattering media
We have recently shown that the use of broadband wavefront shaping to refocus light through scattering media permits to conserve time and polarization properties so as to performe nonlinear optical imaging (second harmonic generation) and polarized imaging.
Highlights:
- Nonlinear imaging through a scattering medium.
H. B. De Aguiar, S. Gigan, S. Brasselet
Enhanced nonlinear imaging through scattering media using transmission matrix based wavefront shaping
Phys. Rev. A 94, 043830 (2016) - Polarized recovery by broadband wavefront shaping through a scattering medium
H.B. De Aguiar, S. Gigan, S. Brasselet
Polarization recovery through scattering media
Sci. Adv. accepted (2017), arXiv:1511.02347
Funding
