Polarized microscopy

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from single molecules to tissues

Principal investigators : Sophie Brasselet

keywords : polarized fluorescence, polarized super-resolution imaging, polarized nonlinear microscopy

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. The methodology is also applied for the investigation of nanostructures, either dielectric or metallic.

Polarized fluorescence microscopy

We develop polarization resolved fluorescence microscopy methods (‘polarimetry’) which are able to probe for each pixel of an image, the degree of orientation of fluorophores attached to proteins of interest. This approach has been successfully applied to probing cell membranes lipid or actin filaments organization, and can be extended to the study of the orientational modifications of membrane proteins for instance. Probing molecular organization has a 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. A major part of this topic is developed today in collaboration with Manos Mavrakis (MOSAIC team), who studies cytoskeletal proteins such as actin and septin.

3D polarized super-resolution fluorescence imaging

We have scaled down polarized fluorescence microscopy at the single molecule scale, in methods which are compatible with super resolution imaging. We have shown the possiblility to measure both mean orientation and the orientational flexibility of fluorophores that are linked to proteins and at the same time 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. This approach has been applied to organized filaments such as dsDNA, actin stress fibers, microtubules. Super-resolution orientation images are reconstructed using polarized STORM (Stochastic Optical Reconstruction Microscopy) and we are extending this approach to other approaches such as PALM or PAINT. We are now extending the previous approaches (limited to 2D projections of the orientational information, to 3D, using polarization splitting as well as point spread function (PSF) engineering approaches. We have in particular shown, in collaboration with Miguel Alonso (MOSAIC team), the possibility to encode in a polarized PSF shape, the orientation, wobbling extend, as well as 3D position of single molecules in a single shot. This PSF engineering is obtained using a spatially birefringent phase plate engineered at Rochester University USA (Tom Brown), which has shown great applications in polarimetry. These appraches open new prospective to super resolution imaging where structural and conformational information can be gained in addition to localization.

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. We are currently developing schemes that allow polarized nonlinear imaging to reach fast imaging rates, either by fast polarization tuning, or via the use of projection on circular polarization combinations. We are also studying the possibilities to develop optical schemes capable to measure the orientation of molecular distributions in 3D, an information which is important in complex tissues. All together these approaches should permit to better quantify molecular order in live and in 3D in tissues related to the study of diseases such as neurodegenerative diseases or cancer.

Polarized microscopy techniques

Fluorescence and nonlinear polarimetry, based on polarization tuning :

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

Polarized super resolution fluorescence imaging :

  • Polarized STORM microscopy
  • 3D polarized STORM microscopy using polarization splitting or PSF engineering

Circular polarization imaging :

  • Nonlinear scanning circularly polarized microscopy (SHG, CARS)



J.-Y. Wang, J.C. Mansfield, S. Brasselet, C. Vergari, J.R. Meakin, C.P. Winlove.
Micro-Mechanical Damages of Needle Puncture on Bovine Annulus Fibrosus Fibrils Studies using 1 Polarisation-Resolved Second Harmonic Generation(P-SHG) Microscopy.
Journal of the Mechanical Behavior of Biomedical Materials (2021)


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).
arXiv:1907.05828, Nat. Communications 11 (1) (2020)
DOI : 10.1038/s41467-020-19064-6


N. Mazumder, N K. Balla, G-Y Zhuo, Y V. Kistenev, R Kumar, F-J Kao, S Brasselet, V V. Nikolaev, N A. Krivova.
Label-free Nonlinear Multimodal Optical Microscopy – Basics, Development and Applications,
Frontiers in Physics, Review, 2019
DOI : 10.3389/fphy.2019.00170 (2019)

M.A. Juanes, D. Isnardon, A. Badache, S. Brasselet, M. Mavrakis, B. Goode,
The role of APC-mediated actin assembly in microtubule capture and focal adhesion turnover,
J. Cell Bio. (2019)
DOI : 10.1083/jcb.201904165

J. Rouxel, H. Shen, N. Nguyen, S. Brasselet, T. Toury.
Enhanced second harmonic generation of gold nanostars : optimizing multipolar radiation to improve nonlinear properties,
Opt. Express 27(4), 5620-5640 (2019)
DOI : 10.1364/OE.27.005620

C. Rendon-Barraza, F. Timpu, R. Grange, S. Brasselet.
Crystalline heterogeneity in single ferroelectric nanocrystals revealed by polarized nonlinear microscopy.
Sci. Reports 9, 1670 (2019)
DOI : 10.1038/s41598-018-38229-4

J. Mansfield, V. Mandalia, A. Toms, C. P. Winlove, S. Brasselet.
Collagen reorganization in cartilage under strain probed by polarization sensitive second harmonic generation microscopy
Journal of the Royal Society Interface 16 (150), pp.20180611 (2019)
DOI : 10.1098/rsif.2018.0611

S. Brasselet
Polarized Microscopy in the Life Sciences.
Optics&Photonics News (2019)


N. Chouaki-Benmansour, K. Ruminski, A.-M. Sartre, M.-C. Phelipot, A. Salles, E. Bergot, A. Wu, G. Chicanne, M. Fallet, S. Brustlein, C. Billaudeau, A. Formisano, S. Mailfert, B. Payrastre, D. Marguet, S. Brasselet, Y. Hamon, H.T. He
Phosphatidylinositol 4,5-bisphosphate regulates the TCR/CD3 complex membrane dynamics and activation,
Sci. Reports 8, 4966 (2018)

N. K. Balla, M. O’Brien, N. McEvoy, G. S. Duesberg, H. Rigneault, S. Brasselet, D. McCloskey.
Effects of Excitonic Resonance on Second and Third Order Nonlinear Scattering from Few-Layer MoS2.
ACS Photonics , 5 (4), pp 1235–1240 (2018)
DOI : 10.1021/acsphotonics.7b00912

O. Loison, M. Weitkunat, A. Kaya-Çopur, C. Nascimento Alves, T. Matzat, M. L. Spletter, S. Luschnig, S. Brasselet, P.-F. Lenne and F. Schnorrer,
Polarization resolved microscopy reveals a muscle myosin motor independent mechanism of molecular actin ordering during sarcomere maturation.
PLoS Biol 16(4) : e2004718 (2018)
DOI : 10.1371/journal.pbio.2004718


H. A. Shaban, C. A. Valades-Cruz, J. Savatier, S. Brasselet,
Polarized super-resolution structural imaging inside amyloid fibrils using Thioflavine T,
Scientific Reports 7 : 12482 (2017)

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. 113 (7), p1520–1530 (2017).
DOI : 10.1016/j.bpj.2017.07.033

H.B. De Aguiar, S. Gigan, S. Brasselet,
Polarization recovery through scattering media,
Science Advances 3 (9), e1600743 ; (2017),
DOI : 10.1126/sciadv.1600743

C. Cleff, H. Rigneault, S. Brasselet, J. Duboisset,
Nonlinear optical susceptibility described with a spherical formalism, applied to coherent anti-Stokes Raman scattering,
Phys. Rev. A 96, 013851 (2017).
DOI : 10.1103/PhysRevA.94.059902

M. Hofer, N.K. Balla, S. Brasselet,
High speed polarization resolved Coherent Raman Scattering imaging,
Optica 4 (7), pp. 795-801 (2017).
DOI : 10.1364/OPTICA.4.000795.

Highlighted in Biophotonics.World (https://www.biophotonics.world/magazine/article/181/new-imaging-technique-able-to-watch-molecular-dynamics-of-neurodegenerative-diseases) and OSA news release (http://www.osa.org/en-us/about_osa/newsroom/news_releases/2017/new_imaging_technique_fast_enough_to_watch_molecul/)

X. Wang, F. Yang, J. Yin, P. Ferrand, S. Brasselet,
Quantifying the polarization properties of non-depolarizing optical elements with virtual distorting elements,
Applied Optics 56 (10) :2589 (2017)

N.K. Balla, C. Rendon-Barraza, L.M. Hoang, P. Karpinski, E. Bermudez, S. Brasselet,
Polarized nonlinear nanoscopy of metal nanostructures,
ACS Photonics 4 (2), pp 292–301 (2017).


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)


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


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)

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)