Extending the capabilities of vectorial ptychography to circular-polarizing materials such as cholesteric liquid crystals.

The problem of imaging materials with circular-polarization properties is discussed within the framework of vectorial ptychography. We demonstrate, both theoretically and numerically, that using linear polarizations to investigate such materials compromises the unicity of the solution provided by this computational method. To overcome this limitation, an improved measurement approach is proposed, which involves specific combinations of elliptical polarizations. The effectiveness of this strategy is demonstrated by numerical simulations and experimental measurements on cholesteric liquid crystal films, which possess unique polarization properties. With the help of Pauli matrices algebra, our results highlight the technique's ability to discern between the different types of circular polarizers, uniform vs. non-uniform, and determine their handedness.

Sub-micrometric spatial distribution of amorphous and crystalline carbonates in biogenic crystals using coherent Raman microscopy.

In living organisms, calcium carbonate biomineralization combines complex bio-controlled physical and chemical processes to produce crystalline hierarchical hard tissues (usually calcite or aragonite) typically from an amorphous precursor phase. Understanding the nature of the successive transient amorphous phases potentially involved in the amorphous-to-crystalline transition requires characterization tools, which are able to provide a spatial and spectroscopic analysis of the biomineral structure. In this work, we present a highly sensitive coherent Raman microscopy approach, which allows one to image molecular bond concentrations in post mortem shells and living animals, by exploiting the vibrational signature of the different carbonates compounds. To this end, we target the ν1 calcium carbonate vibration mode and produce spatially and spectroscopically resolved images of the shell border of a mollusk shell, the Pinctada margaritifera pearl oyster. A novel approach is further presented to efficiently compare the amount of amorphous carbonate with respect to its crystalline counterpart. Finally, the whole microscopy method is used to image in vivo the shell border and demonstrate the feasibility and the reproducibility of the technique. These findings open chemical imaging perspectives for the study of biogenic and bio-inspired crystals.

Amorphous-to-crystal transition in the layer-by-layer growth of bivalve shell prisms.

Biomineralization integrates complex physical and chemical processes bio-controlled by the living organisms through ionic concentration regulation and organic molecules production. It allows tuning the structural, optical and mechanical properties of hard tissues during ambient-condition crystallisation, motivating a deeper understanding of the underlying processes. By combining state-of-the-art optical and X-ray microscopy methods, we investigated early-mineralized calcareous units from two bivalve species, Pinctada margaritifera and Pinna nobilis, revealing chemical and crystallographic structural insights. In these calcite units, we observed ring-like structural features correlated with a lack of calcite and an increase of amorphous calcium carbonate and proteins contents. The rings also correspond to a larger crystalline disorder and a larger strain level. Based on these observations, we propose a temporal biomineralization cycle, initiated by the production of an amorphous precursor layer, which further crystallizes with a transition front progressing radially from the unit centre, while the organics are expelled towards the prism edge. Simultaneously, along the shell thickness, the growth occurs following a layer-by-layer mode. These findings open biomimetic perspectives for the design of refined crystalline materials. STATEMENT OF SIGNIFICANCE: Calcareous biominerals are amongst the most present forms of biominerals. They exhibit astonishing structural, optical and mechanical properties while being formed at ambient synthesis conditions from ubiquitous ions, motivating the deep understanding of biomineralization. Here, we unveil the first formation steps involved in the biomineralization cycle of prismatic units of two bivalve species by applying a new multi-modal non-destructive characterization approach, sensitive to chemical and crystalline properties. The observations of structural features in mineralized units of different ages allowed the derivation of a temporal sequence for prism biomineralization, involving an amorphous precursor, a radial crystallisation front and a layer-by-layer sequence. Beyond these chemical and physical findings, the herein introduced multi-modal approach is highly relevant to other biominerals and bio-inspired studies.

Broadband decoupling of intensity and polarization with vectorial Fourier metasurfaces.

Intensity and polarization are two fundamental components of light. Independent control of them is of tremendous interest in many applications. In this paper, we propose a general vectorial encryption method, which enables arbitrary far-field light distribution with the local polarization, including orientations and ellipticities, decoupling intensity from polarization across a broad bandwidth using geometric phase metasurfaces. By revamping the well-known iterative Fourier transform algorithm, we propose "à la carte" design of far-field intensity and polarization distribution with vectorial Fourier metasurfaces. A series of non-conventional vectorial field distribution, mimicking cylindrical vector beams in the sense that they share the same intensity profile but with different polarization distribution and a speckled phase distribution, is demonstrated. Vectorial Fourier optical metasurfaces may enable important applications in the area of complex light beam generation, secure optical data storage, steganography and optical communications.

Reference-free quantitative microscopic imaging of coherent arbitrary vectorial light beams.

Precise spatial characterization of vectorial beams is crucial for many advanced optical experiments, but challenging when wavefront and polarization features are involved together. Here we propose a reference-free method aimed at extracting the map of the complex-amplitude components of any coherent beam at an optical-microscopy resolution. Our method exploits recent advances in ptychographic imaging approaches. We emphasize its versatility by reconstructing successfully various experimental vectorial beams including polarization and phase vortices, the exit field of a multicore fiber and a speckle pattern.

Ptychography retrieval of fully polarized holograms from geometric-phase metasurfaces.

Controlling light properties with diffractive planar elements requires full-polarization channels and accurate reconstruction of optical signal for real applications. Here, we present a general method that enables wavefront shaping with arbitrary output polarization by encoding both phase and polarization information into pixelated metasurfaces. We apply this concept to convert an input plane wave with linear polarization to a holographic image with arbitrary spatial output polarization. A vectorial ptychography technique is introduced for mapping the Jones matrix to monitor the reconstructed metasurface output field and to compute the full polarization properties of the vectorial far field patterns, confirming that pixelated interfaces can deflect vectorial images to desired directions for accurate targeting and wavefront shaping. Multiplexing pixelated deflectors that address different polarizations have been integrated into a shared aperture to display several arbitrary polarized images, leading to promising new applications in vector beam generation, full color display and augmented/virtual reality imaging.

Joint estimation of object and probes in vectorial ptychography.

Vectorial ptychography has been recently introduced to reconstruct the Jones matrix of an anisotropic object by means of series of ptychographic measurements performed using a set of polarized illumination probes in conjugation with various analyzers. So far, the probes were assumed to be completely known (amplitude, wavefront, state of polarization), which is rarely the case in practice. Here we address the issue of the joint estimating of the set of polarized illumination probes together with the Jones matrix of an anisotropic object in vectorial ptychography. We propose an algorithm based on a conjugate gradient strategy. Experimental results are reported, showing an improvement on the object estimate, in addition to a precise reconstruction of the probes.

Quantitative imaging of anisotropic material properties with vectorial ptychography.

Following the recent establishment of the formalism of vectorial ptychography [Opt. Lett.40, 5144 (2015)OPLEDP0146-959210.1364/OL.40.005144], first measurements, to the best of our knowledge, are reported in the optical range, demonstrating the capability of the proposed method to map the four parameters of the Jones matrix of an anisotropic specimen, and therefore to quantify a wide range of optical material properties, including power transmittance, optical path difference, diattenuation, retardance, and fast-axis orientation.

Lipid Order Degradation in Autoimmune Demyelination Probed by Polarized Coherent Raman Microscopy.

Myelin around axons is currently widely studied by structural analyses and large-scale imaging techniques, with the goal to decipher its critical role in neuronal protection. Although there is strong evidence that in myelin, lipid composition, and lipid membrane morphology are affected during the progression of neurodegenerative diseases, there is no quantitative method yet to report its ultrastructure in tissues at both molecular and macroscopic levels, in conditions potentially compatible with in vivo observations. In this work, we study and quantify the molecular order of lipids in myelin at subdiffraction scales, using label-free polarization-resolved coherent anti-Stokes Raman, which exploits coherent anti-Stokes Raman sensitivity to coupling between light polarization and oriented molecular vibrational bonds. Importantly, the method does not use any a priori parameters in the sample such as lipid type, orientational organization, and composition. We show that lipid molecular order of myelin in the mouse spinal cord is significantly reduced throughout the progression of experimental autoimmune encephalomyelitis, a model for multiple sclerosis, even in myelin regions that appear morphologically unaffected. This technique permits us to unravel molecular-scale perturbations of lipid layers at an early stage of the demyelination progression, whereas the membrane architecture at the mesoscopic scale (here ∼100 nm) seems much less affected. Such information cannot be brought by pure morphological observation and, to our knowledge, brings a new perspective to molecular-scale understanding of neurodegenerative diseases.

Quantifying the polarization properties of non-depolarizing optical elements with virtual distorting elements: publisher's note.

This publisher's note amends the author list and Acknowledgments in Appl. Opt.56, 2589 (2017)APOPAI0003-693510.1364/AO.56.002589.

Direct imaging of molecular symmetry by coherent anti-stokes Raman scattering.

Nonlinear optical methods, such as coherent anti-Stokes Raman scattering and stimulated Raman scattering, are able to perform label-free imaging, with chemical bonds specificity. Here we demonstrate that the use of circularly polarized light allows to retrieve not only the chemical nature but also the symmetry of the probed sample, in a single measurement. Our symmetry-resolved scheme offers simple access to the local organization of vibrational bonds and as a result provides enhanced image contrast for anisotropic samples, as well as an improved chemical selectivity. We quantify the local organization of vibrational bonds on crystalline and biological samples, thus providing information not accessible by spontaneous Raman and stimulated Raman scattering techniques. This work stands for a symmetry-resolved contrast in vibrational microscopy, with potential application in biological diagnostic.

Coupling Emitters and Silver Nanowires to Achieve Long-Range Plasmon-Mediated Fluorescence Energy Transfer.

The development of quantum plasmonic circuitry requires efficient coupling between quantum emitters and plasmonic waveguides. A major experimental challenge is to simultaneously maximize the surface plasmon propagation length, the coupling efficiency into the plasmonic mode, and the Purcell factor. Addressing this challenge is also the key to enabling long-range energy transfer between quantum nanoemitters. Here, we use a dual-beam scanning confocal microscope to carefully investigate the interactions between fluorescent nanoparticles and surface plasmons on single-crystalline silver nanowires. By exciting the fluorescent nanoparticles via nanowire surface plasmons, we maximize the light-matter interactions and reach coupling efficiencies up to 44% together with 24× lifetime reduction and 4.1 µm propagation lengths. This improved optical performance enables the demonstration of long-range plasmon-mediated fluorescence energy transfer between two nanoparticles separated by micrometer distance. Our results provide guidelines toward practical realizations of efficient long-range fluorescence energy transfer for integrated plasmonics and quantum nano-optics.

Ptychography in anisotropic media.

This letter describes ptychography in the context of polarized light probing anisotropic specimen (i.e., showing properties of birefringence and/or diattenuation). We established an optimization strategy using a vectorial formalism. We propose a measurement scheme using a set of linearly polarized probes and linear polarization analyzers, which allows the retrieval of the full anisotropy map of the specimen.

Molecular orientational order probed by coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy: a spectral comparative study.

We investigate how to extract information on the orientational order of molecular bonds in biological samples from polarized coherent anti-Stokes Raman scattering (CARS) and stimulated Raman scattering (SRS) microscopy. Experimentally, the mean orientation of the molecular angular distribution, as well as its second and fourth orders of symmetry, are estimated by monitoring intensity signals under a varying incident polarization. We provide a generic method of analysis of polarized signals in both CARS and SRS contrasts, and apply it to imaging of lipid bonds' orientational order in multilamellar vesicles. A comparison of the two contrasts in the lipid region around 3000 cm(-1) shows that while SRS allows retrieving pure molecular order information, CARS is generally tainted by a bias from the nonresonant contribution.

Fiber-based ultrashort pulse delivery for nonlinear imaging using high-energy solitons.

We present an approach for fiber delivery of femtosecond pulses relying on pulse breakup and soliton self-frequency shift in a custom-made solid-core photonic bandgap fiber. In this scheme, the fiber properties themselves ensure that a powerful Fourier-transform-limited pulse is emitted at the fiber output, hence doing away with the need for complex precompensation and enabling tunability of the excitation. We report high-energy soliton excitation for two-photon fluorescence microspectroscopy over a 100-nm range and multimodal nonlinear imaging on biological samples.

Ultimate use of two-photon fluorescence microscopy to map orientational behavior of fluorophores.

The orientational distribution of fluorophores is an important reporter of the structure and function of their molecular environment. Although this distribution affects the fluorescence signal under polarized-light excitation, its retrieval is limited to a small number of parameters. Because of this limitation, the need for a geometrical model (cone, Gaussian, etc.) to effect such retrieval is often invoked. In this work, using a symmetry decomposition of the distribution function of the fluorescent molecules, we show that polarized two-photon fluorescence based on tunable linear dichroism allows for the retrieval of this distribution with reasonable fidelity and without invoking either an a priori knowledge of the system to be investigated or a geometrical model. We establish the optimal level of detail to which any distribution can be retrieved using this technique. As applied to artificial lipid vesicles and cell membranes, the ability of this method to identify and quantify specific structural properties that complement the more traditional molecular-order information is demonstrated. In particular, we analyze situations that give access to the sharpness of the angular constraint, and to the evidence of an isotropic population of fluorophores within the focal volume encompassing the membrane. Moreover, this technique has the potential to address complex situations such as the distribution of a tethered membrane protein label in an ordered environment.

Mapping the local organization of cell membranes using excitation-polarization-resolved confocal fluorescence microscopy.

Fluorescence anisotropy and linear dichroism imaging have been widely used for imaging biomolecular orientational distributions in protein aggregates, fibrillar structures of cells, and cell membranes. However, these techniques do not give access to complete orientational order information in a whole image, because their use is limited to parts of the sample where the average orientation of molecules is known a priori. Fluorescence anisotropy is also highly sensitive to depolarization mechanisms such as those induced by fluorescence energy transfer. A fully excitation-polarization-resolved fluorescence microscopy imaging that relies on the use of a tunable incident polarization and a nonpolarized detection is able to circumvent these limitations. We have developed such a technique in confocal epifluorescence microscopy, giving access to new regions of study in the complex and heterogeneous molecular organization of cell membranes. Using this technique, we demonstrate morphological changes at the subdiffraction scale in labeled COS-7 cell membranes whose cytoskeleton is perturbed. Molecular orientational order is also seen to be affected by cholesterol depletion, reflecting the strong interplay between lipid-packing regions and their nearby cytoskeleton. This noninvasive optical technique can reveal local organization in cell membranes when used as a complement to existing methods such as generalized polarization.

High frame-rate fluorescence confocal angle-resolved linear dichroism microscopy.

Angle-resolved linear dichroism is a recent technique that exploits images recorded using an illumination field whose polarization angle is sequentially rotated during acquisition. It allows to retrieve orientation information of the fluorescent molecules, namely the average orientation angle and the amplitude of the fluctuations around this average. In order to boost up the acquisition speed without sacrificing the axial sectioning, we propose to combine a spinning disk confocal excitation scheme together with an electrooptical polarization switching and a camera acquisition. The polarization distortions induced when passing through the spinning disk system have been quantified and effectively compensated. The signal to noise features of the camera have been analyzed in detail so that the precision of the method can be quantified. The technique has been successfully tested on giant unilamellar vesicles and on living cells labeled with different fluorescent lipid probes, DiIC18 and di-8-ANEPPQ. It was able to acquire precise orientation images at full frame rates in the range of a second, ultimately limited by the fluorophore brightness and the camera sensitivity.

Thioflavine-T and Congo Red reveal the polymorphism of insulin amyloid fibrils when probed by polarization-resolved fluorescence microscopy.

Amyloid fibrils are protein misfolding structures that involve a ß-sheet structure and are associated with the pathologies of various neurodegenerative diseases. Here we show that Thioflavine-T and Congo Red, two major dyes used to image fibrils by fluorescence assays, can provide deep structural information when probed by means of polarization-resolved fluorescence microscopy. Unlike fluorescence anisotropy or fluorescence detected linear dichroism imaging, this technique allows to retrieve simultaneously both mean orientation and orientation dispersion of the dye, used here as a reporter of the fibril structure. We have observed that insulin amyloid fibrils exhibit a homogeneous behavior over the fibrils' length, confirming their structural uniformity. In addition, these results reveal the existence of various structures among the observed fibrils' population, in spite of a similar aspect when imaged with conventional fluorescence microscopy. This optical nondestructive technique opens perspectives for in vivo structural analyses or high throughput screening.

Microscopic structural study of collagen aging in isolated fibrils using polarized second harmonic generation.

Polarization resolved second harmonic generation (PSHG) is developed to study, at the microscopic scale, the impact of aging on the structure of type I collagen fibrils in two-dimensional coatings. A ribose-glycated collagen is also used to mimic tissue glycation usually described as an indicator of aging. PSHG images are analyzed using a generic approach of the molecular disorder information in collagen fibrils, revealing significant changes upon aging, with a direct correlation between molecular disorder and fibril diameters.