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Polarized microscopy

from single molecules to tissues

Principal investigators : Sophie Brasselet, Julien Duboisset, Julien Savatier

keywords : polarized fluorescence, polarized nonlinear microscopy, tunable linear dichroism, anisotropy, polarized super-resolution imaging

Polarization is an important property of light that is often ignored in light-matter interaction, in particular for bio-imaging applications. We develop methodologies for optical microscopy that exploit the properties of light polarization by a control of the incident or detected polarization states. This approach allows retrieving molecular orientational information in biological media, from complex bio-molecular assemblies down to single molecules.

Polarized fluorescence microscopy

We have performed studies on the orientational organization of lipid probes and labeled proteins within the plasma membrane and actin filaments in fixed cells and contractile rings in Drosophila embryos. This information on molecular organization has great potential for providing a new means of visualizing the reorganization of cytoskeletal filaments for instance during morphogenetic events in real time at the molecular scale, to elucidate their contribution to mechanical properties of cells and tissues, as well as to decipher the role of regulatory proteins.

This experimental approach is supported by intensive research on the optimization of signal treatment, in order to guaranty optimal estimation of molecular order parameters depending on a given setting of incident/detected polarizations. This work is carried out in collaboration with PHYTI (I. Fresnel) to evaluate the Cramer Rao bond properties in different configurations used in our experiments.

Polarized super-resolution imaging

Fluorescence polarized microscopy is scaled down to the single molecule scale, in order to (1) get more information on the orientational flexibility of the fluorophores that are linked to proteins, (2) reconstruct super-resolution orientation images using their localization information. Orientational flexibility is an important parameter to quantify to be able to retrieve realistic structural information on the labeled bio-molecules. A wide field polarized fluorescence experiment is developed that can give both mean orientation and wobbling extent for each single molecule attached to filament structures in vitro and in cells. This approach is applied to organized filaments such as dsDNA, actin stress fibers, microtubules. Super-resolution orientation images are reconstructed using polarized dSTORM (direct Stochastic Optical Reconstruction Microscopy)

Polarized non-linear imaging

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). 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 structure changes, in mice spinal cord tissues.

Two developments are currently actively studied, on the effect of scattering on polarization properties in biological tissues, and on the possibility to perform live molecular order imaging avoiding polarization tuning, by the use of circular polarizations.

Polarized microscopy techniques

Tunable linear dichroism (incident polarization tuning) imaging

  • Confocal fluorescence microscopy
  • Spinning disk fluorescence microscopy (live polarization imaging)
  • Nonlinear scanning polarized microscopy (2-photon fluorescence, SHG, CARS, SRS)

Anisotropy imaging (detected polarization tuning)

  • Polarization dSTORM microscopy

Circular polarization imaging

  • Nonlinear scanning circularly polarized microscopy (SHG, CARS)

Recent publications

H. B. de Aguiar, P. Gasecka and S. Brasselet
Quantitative analysis of light scattering in polarization-resolved nonlinear microscopy
Opt. Express 23 (7), pp. 8960-8973 (2015)

J. Duboisset, P. Berto, P. Gasecka, F..Z. Bioud, P. Ferrand, H. Rigneault , S. Brasselet
Molecular orientational order probed by coherent anti-Stokes Raman scattering (CARS) and stimulated Raman Scattering (SRS) microscopy : a spectral comparative study
J. Phys. Chem. B 119 (7), pp 3242–3249 (2015)

V. Wasik, P. Réfrégier, M. Roche and S. Brasselet
Precision of polarization-resolved second harmonic generation microscopy limited by photon noise for samples with cylindrical symmetry
JOSA A 32(8) 1437-1445 (2015)

J. Duboisset, H. Rigneault, S. Brasselet
‘Filtering of matter symmetry properties by circularly polarized nonlinear optics
Phys. Rev. A 90, 063827 (2014)

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)

F.-Z. Bioud, P. Gasecka, P. Ferrand, H. Rigneault, J. Duboisset, and S. Brasselet
Structure of molecular packing probed by polarization-resolved nonlinear four-wave mixing and coherent anti-Stokes Raman scattering microscopy
Phys. Rev. A 89, 013836 (2014)

P. Ferrand, P. Gasecka, A. Kress, X. Wang, F.-Z. Bioud, J. Duboisset, S. Brasselet
Ultimate use of two-photon fluorescence microscopy to map fluorophores orientational behavior
Biophys. J. 106 2330–2339 (2014)

S. Brasselet, P. Ferrand, A. Kress, X. Wang, H. Ranchon, A. Gasecka
Imaging Molecular Order in Cell Membranes by Polarization-Resolved Fluorescence Microscopy
Y. Mély and G. Duportail (eds.), Fluorescent Methods to Study Biological Membranes, Springer Series Fluorescence
Springer-Verlag Berlin Heidelberg 2012, 13, 311-338 (2013)

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)

H. Shen, N. Nguyen, D. Gachet, V. Maillard, T. Toury, S. Brasselet
Nanoscale optical properties of metal nanoparticles probed by Second Harmonic Generation microscopy
Opt. Express 21 (10), pp.12318-12326 (2013)

X. Wang, A. Kress, S. Brasselet, P. Ferrand
High frame-rate confocal angular resolved linear dichroism fluorescence microscopy
Rev. Sc. Instr. Rev. Sci. Instrum. 84, 053708 (2013)

J. Duboisset, P. Ferrand, H. Wei, X. Wang, H. Rigneault, S. Brasselet
Thioflavine-T and Congo Red Reveal the Polymorphism of Insulin Amyloid Fibrils when Probed by Polarization-Resolved Fluorescence Microscopy
J. Phys. Chem. B, 2013, 117 (3), pp 784–788

P. Refregier, M. Roche, J. Duboisset, S. Brasselet
Precision increase with two orthogonal analyzers in polarization resolved second harmonic generation microscopy
Opt. Lett. 37 (20), pp.4173-4175 (2012)

D. Ait-Belkacem, M. Guilbert, M. Roche, J. Duboisset, P. Ferrand, G. Sockalingum, P. Jeannesson, and S. Brasselet
Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation
J. Biomed. Opt. 17, 080506 (2012)

F. Munhoz, H. Rigneault, S. Brasselet
Polarization–resolved four-wave mixing for structural imaging in thick tissues
J. Opt. Soc. Am. B 29 (6), pp.1541-1550 (2012)

J. Duboisset, Dora Aït-Belkacem, Muriel Roche, Hervé Rigneault, Sophie Brasselet
Generic model of the molecular orientational distribution probed by polarization resolved Second Harmonic Generation
Phys. Rev. A. 85, 043829-38 (2012)

A. Gasecka, P. Tauc, A. Bentley, S. Brasselet
Investigation of Molecular and Protein Crystals by Three Photon Polarization Resolved Microscopy
Phys Rev Lett 108, 263901-05 (2012)

F. Munhoz, S. Brustlein, R. Hostein, P. Berto, S. Brasselet and H. Rigneault
Polarization resolved stimulated Raman scattering : probing depolarization ratios of liquids
Journal of Raman Spectroscopy 43 (3), 419–424 (2012)

S. Monneret, S. Brasselet
Advanced microscopy techniques for biological imaging
Int. J. Nanotechnol., Vol. 9 (3–7), 548-566 (2012)

A. Kress, P. Ferrand, H. Rigneault, T. Trombik, H.-T. He, D. Marguet, S. Brasselet
Probing orientational behavior of MHC Class I protein and lipid probes in cell membranes by fluorescence polarization-resolved imaging
Biophys. J. 101, pp. 468–476 (2011)

P. Refregier, M. Roche, S. Brasselet
Precision analysis in polarization-resolved second harmonic generation microscopy
Opt. Lett. Vol. 36 (11), pp. 2149-2151 (2011)

S. Brasselet
Polarization resolved nonlinear microscopy : application to structural molecular and biological imaging
Advances in Optics and Photonics 3, pp. 205–271 (2011)

F. Munhoz, H. Rigneault, S. Brasselet
High Order Symmetry Structural Properties of Vibrational Resonances Using Multiple-Field Polarization Coherent Anti-Stokes Raman Spectroscopy Microscopy
Phys Rev Lett. 105, 123903 (2010)

D. Aït-Belkacem, A. Gasecka, F. Munhoz, S. Brustlein, and S. Brasselet
Influence of birefringence on polarization resolved nonlinear microscopy and collagen SHG structural imaging
Opt. Express 18 (14) 14859-14870 (2010)

S. Brasselet
Second Harmonic Generation microscopy in molecular crystalline nano-objects
Nonlinear Optics, Quantum Optics NLOQO, 40 (1-4), pp 83-94 (2010)

A. Gasecka, L-Q. Dieu, D. Bruehviler, S. Brasselet
Probing molecular order in zeolite L inclusion compounds using two-photon fluorescence polarimetric microscopy
J. Phys. Chem. B 114 (12), pp 4192–4198 (2010)

P. Schön, M. Behrndt, D. Ait-Belkacem, H. Rigneault, S. Brasselet
Polarization and Phase Pulse Shaping applied to Structural Contrast in Nonlinear Microscopy Imaging
Phys. Rev. A 81, 013809 (2010)

F. Munhoz, S. Brustlein, D. Gachet, F. Billard, S. Brasselet, H. Rigneault
Raman depolarization ratio of liquids probed by linear polarization Coherent Anti-Stokes Raman spectroscopy
Journal of Raman Spectroscopy, vol. 40, (7), 775-780 (2009)

A. Gasecka, T.-J. Han, C. Favard, B.R. Cho, S. Brasselet
Quantitative imaging of molecular order in lipid membranes using two-photon fuorescence polarimetry
Biophys J. 97 (10) 2854-2862 (2009)