Clément Fallet

Polarimetric imaging of uterine cervix: a case study

We present a preliminary investigation of macroscopic polarimetric imaging of uterine cervix. Orthogonal state contrast (OSC) images of healthy and anomalous cervices have been taken in vivo at 550 nm. Four ex vivo cervix samples have been studied in full Muller polarimetry, at 550 nm and 700 nm, and characterized in detail by standard pathology. One sample was totally healthy, another one carried CIN lesions at very early stage (CIN1) in its visible exocervical region, while for the other two samples more advanced (CIN3) lesions were present, together with visible glandular epithelium (ectropion). Significant birefringence has been observed in the healthy regions of all six samples, both in vivo and ex vivo. Standard treatments of the Mueller images of the ex vivo samples allowed to quantify both retardation and depolarization. Retardation reached 60° in healthy regions, and disappeared in the anomalous regions of the other three ex vivo samples. The depolarization power was largest in healthy regions, and lower in CINs and ectropion. Possible origins of the observed effects are briefly discussed.

Multispectral Mueller polarimetric imaging detecting residual cancer and cancer regression after neoadjuvant treatment for colorectal carcinomas

Abstract. This work is devoted to a first exploration of Mueller polarimetric imaging for the detection of residual cancer after neoadjuvant treatment for the rectum. Three samples of colorectal carcinomas treated by radiochemotherapy together with one untreated sample are analyzed ex vivo before fixation in formalin by using a multispectral Mueller polarimetric imaging system operated from 500 to 700 nm. The Mueller images, analyzed using the Lu-Chipmann decomposition, show negligible diattenuation and retardation. The nonirradiated rectum exhibits a variation of depolarization with cancer evolution stage. At all wavelengths on irradiated samples, the contrast between the footprint of the initial tumor and surrounding healthy tissue is found to be much smaller for complete tumor regression than when a residual tumor is present, even at volume fractions of the order of 5%. This high sensitivity is attributed to the modification of stromal collagen induced by the cancer. The depolarization contrast between treated cancer and healthy tissue is found to increase monotonously with the volume fraction of residual cancer in the red part of the spectrum. Polarimetric imaging is a promising technique for detecting short-time small residual cancers, which is valuable information for pathological diagnosis and patient management by clinicians.

Ex vivo photometric and polarimetric multilayer characterization of human healthy colon by multispectral Mueller imaging

Healthy human colon samples were analyzed ex vivo with a multispectral imaging Mueller polarimeter operating from 500 to 700 nm in a backscattering configuration with diffuse light illumination impinging on the innermost tissue layer, the mucosa. The intensity and polarimetric responses were taken on whole tissues first and after progressive exfoliation of the outer layers afterwards. Moreover, these measurements were carried out with two different substrates (one bright and the other dark) successively placed beneath each sample, allowing a reasonably accurate evaluation of the contributions to the overall backscattered light by the various layers. For the shorter investigated wavelengths (500 to 550 nm) the major contribution comes from mucosa and submucosa, while for the longer wavelengths (650 to 700 nm) muscular tissue and fat also contribute significantly. The depolarization has also been studied and is found to be stronger in the red part of the spectrum, mainly due to the highly depolarizing power of the muscular and fat layers.

Experimental evidence for naturally occurring nondiagonal depolarizers

We report on two Stokes nondiagonalizable Mueller matrices experimentally observed in a biological and in an organic sample. These matrices are examples of naturally occurring nondiagonal depolarizers whose unique property is to preserve the degree of polarization of all but one totally polarized light state. The description of the experimental matrices within the theory of Bragg scattering on cholesteric liquid crystals, as well as their interpretation in physical and structural terms, are likewise addressed.

Conical diffraction illumination opens the way for low phototoxicity super-resolution imaging

We present a new technology for super-resolution fluorescence imaging, based on conical diffraction. Conical diffraction is a linear, singular phenomenon, taking place when a laser beam is diffracted through a biaxial crystal. We use conical diffraction in a thin biaxial crystal to generate illumination patterns that are more compact than the classical Gaussian beam, and use them to generate a super-resolution imaging modality. While there already exist several super-resolution modalities, our technology (biaxial super-resolution: BSR) is distinguished by the unique combination of several performance features. Using BSR super-resolution data are achieved using low light illumination significantly less than required for classical confocal imaging, which makes BSR ideal for live-cell, long-term time-lapse super-resolution imaging. Furthermore, no specific sample preparation is required, and any fluorophore can be used. Perhaps most exciting, improved resolution BSR-imaging resolution enhancement can be achieved with any type of objective no matter the magnification, numerical aperture, working distance, or the absence or presence of immersion medium. In this article, we present the first implementation of BSR modality on a commercial confocal microscope. We acquire and analyze validation data, showing high quality super-resolved images of biological objects, and demonstrate the wide applicability of the technology. We report live-cell super-resolution imaging over a long period, and show that the light dose required for super-resolution imaging is far below the threshold likely to generate phototoxicity.

Experimental implementation and properties of Stokes nondiagonalizable depolarizing Mueller matrices

We report on the experimental realization of a family of depolarizing Mueller matrices that are Stokes nondiagonalizable. We likewise demonstrate a unique characteristic property of Stokes nondiagonalizable matrices consisting in the preservation of the degree of polarization of a single totally polarized input and illustrate it on our experimental example.

Sum decomposition of Mueller-matrix images and spectra of beetle cuticles

Spectral Mueller matrices measured at multiple angles of incidence as well as Mueller matrix images are recorded on the exoskeletons (cuticles) of the scarab beetles Cetonia aurata and Chrysina argenteola. Cetonia aurata is green whereas Chrysina argenteola is gold-colored. When illuminated with natural (unpolarized) light, both species reflect left-handed and near-circularly polarized light originating from helicoidal structures in their cuticles. These structures are referred to as circular Bragg reflectors. For both species the Mueller matrices are found to be nondiagonal depolarizers. The matrices are Cloude decomposed to a sum of non-depolarizing matrices and it is found that the cuticle optical response, in a first approximation can be described as a sum of Mueller matrices from an ideal mirror and an ideal circular polarizer with relative weights determined by the eigenvalues of the covariance matrices of the measured Mueller matrices. The spectral and image decompositions are consistent with each other. A regression-based decomposition of the spectral and image Mueller matrices is also presented whereby the basic optical components are assumed to be a mirror and a circular polarizer as suggested by the Cloude decomposition. The advantage with a regression decomposition compared to a Cloude decomposition is its better stability as the matrices in the decomposition are determined a priori. The origin of the depolarizing features are discussed but from present data it is not possible to conclude whether the two major components, the mirror and the circular polarizer are laterally separated in domains in the cuticle or if the depolarization originates from the intrinsic properties of the helicoidal structure.

Overlay Measurements by Mueller Polarimetry in Back Focal Plane

Angle resolved Mueller polarimetry implemented as polarimetric imaging of a back focal plane of a high NA microscope objective has already demonstrated a good potential for CD metrology. Here we present the experimental and numerical results indicating that this technique may also be competitive for the measurements of overlay error δ. A series of samples of superimposed gratings with well controlled overlay errors have been manufactured and measured with the angle resolved Mueller polarimeter. The overlay targets were 20-μm wide. When the overlay error is δ is equal to 0, absolute values of elements of real 4×4 Mueller matrix M are invariant by matrix transposition. Otherwise this symmetry breaks down. Consequently, we define the following overlay estimator matrix as E = |M| − |M|t. The simulations show that matrix element E14 is the most sensitive to the overlay error. The scalar estimator of E14 was calculated by averaging the pixel values over a specifically chosen mask. This estimator is found to vary linearly with δ for overlay values up to 50 nm. Our technique allows entering small overlay marks (down to 5-μm wide). Only one target measurement is needed for each overlay direction. The actual overlay value can be determined without detailed simulation of the structure provided two calibrated overlay structures are available for each direction.

Conical diffraction as a versatile building block to implement new imaging modalities for superresolution in fluorescence microscopy

We present a new technology for super-resolution fluorescence imaging, based on conical diffraction. Conical diffraction is a linear, singular phenomenon taking place when a polarized beam is diffracted through a biaxial crystal. The illumination patterns generated by conical diffraction are more compact than the classical Gaussian beam; we use them to generate a super-resolution imaging modality. Conical Diffraction Microscopy (CODIM) resolution enhancement can be achieved with any type of objective on any kind of sample preparation and standard fluorophores. Conical diffraction can be used in multiple fashion to create new and disruptive technologies for super-resolution microscopy. This paper will focus on the first one that has been implemented and give a glimpse at what the future of microscopy using conical diffraction could be.

Overlay measurements by Mueller polarimetry in the back focal plane

Angle resolved Mueller polarimetry implemented as polarimetric imaging of the back focal plane of a high NA microscope objective has already demonstrated a good potential for CD metrology1. In this paper we present the experimental and numerical results which indicate that this technique may also be competitive for measurements of the overlay error δ between two gratings at different levels. Series of samples of superimposed gratings with well controlled overlay errors have been manufactured and measured with the angle resolved Mueller polarimeter. The overlay targets were 20 μm wide. When overlay error δ = 0 the absolute value of Mueller matrix elements is invariant by matrix transposition. This symmetry breaks down when δ ≠ 0. As a result, we can define the following overlay estimator matrix: Ε = |Μ | - |Μ |t. The simulations show that matrix element E14 is the most sensitive to the overlay error. In the experiments the scalar estimator of E14 was defined by averaging the pixel values over specifically chosen mask. The scalar estimator is found to vary essentially linearly with δ for the overlay values up to 50 nm. Our technique allows entering quite small overlay marks (down to 5 μm wide). The only one target measurement is needed for each overlay direction. The actual overlay value can be determined without detailed simulation of the structure provided the two calibrated overlay structures are available for each direction.