Swept-wavelength null polarimetry for highly sensitive birefringence laser scanning microscopy.

We have recently demonstrated a high-speed null polarimeter [Opt. Express30, 18889 (2022)10.1364/OE.454193OPEXFF1094-4087] based on passive polarization optics and using a fast swept-wavelength laser source. We report here its implementation in a laser-scanning microscope setup, enabling highly sensitive linear retardance imaging with a pixel dwell time of 10 µs. The instrument is also able to measure light depolarization induced by the sample. Images of biological samples, including cancerous tissue and cells, illustrate its performances.

Systematic errors in a swept-wavelength null polarimeter for weak linear retardance measurements.

We recently developed a high speed null polarimeter [Opt. Express30, 18889 (2022)OPEXFF1094-408710.1364/OE.454193] based on passive polarization optics and a high speed wavelength swept laser source, enabling the measurement of linear retardance with 3.1µd e g/H z resolution within a minimum acquisition time of 10 µs, corresponding to a linear retardation of 8.6×10-9 λ/H z. The counterpart of high sensitivity lies in the systematic errors unlike Mueller polarimeters, which can be calibrated but which are much less sensitive. This paper focuses on the accuracy of this null polarimeter and provides hardware and numerical solutions to improve both linear retardance and azimuth measurements. Experiments and theoretical simulations are carried out to demonstrate the relevancy of these solutions.

Swept-wavelength null polarimeter for high-speed weak anisotropy measurements.

Null-polarimeters provide the best sensitivity to anisotropy measurements and so far have been developed for the detection of small optical activities. This paper revisits null polarimetry through an original configuration based on the concept of spectrally encoded light polarization, in order to measure, with unprecedented speed, either linear or circular retardance with the same degree of sensitivity . Using passive polarization optics and a high speed wavelength swept laser source, the achieved single-pass sensitivity was 55nrad/Hz and 45nrad/Hz for respectively linear and circular retardance considering a minimum acquisition time of 10 µs. Due to its compactness and rapidity, the method could be further implemented in laser scanning microscopes, which should be of great interest for revealing very low anisotropies in biological tissues.

Gastric cancer multicellular spheroid analysis by two-photon microscopy.

Gastric cancer (GC) is highly deadly. Three-dimensional (3D) cancer cell cultures, known as spheroids, better mimic tumor microenvironment (TME) than standard 2D cultures. Cancer-associated fibroblasts (CAF), a major cellular component of TME, promote or restrain cancer cell proliferation, invasion and resistance to drugs. We established spheroids from two human GC cell lines mixed with human primary CAF. Spheroid organization, analyzed by two-photon microscopy, showed CAF in AGS/CAF spheroids clustered in the center, but dispersed throughout in HGT-1/CAF spheroids. Such differences may reflect clonal specificities of GC cell lines and point to the fact that GC should be considered as a highly personalized disease.

Linear diattenuation imaging of biological tissues with near infrared Mueller scanning microscopy.

Among the multitude of optical polarization contrasts that can be observed in complex biological specimens, linear diattenuation (LD) imaging has received little attention. It is indeed challenging to image LD with basic polarizing microscopes because it is often relatively small in comparison with linear retardance (LR). In addition, interpretation of LD images is not straightforward when experiments are conducted in the visible range because LD can be produced by both dichroism and anisotropic scattering. Mueller polarimetry is a powerful implementation of polarization sensing able to differentiate and measure the anisotropies of specimens. In this article, near infrared transmission Mueller scanning microscopy is used to image LD in thin biological specimen sections made of various proteins with unprecedented resolution and sensitivity. The near infrared spectral range makes it possible to lower the contribution of dichroism to the total linear diattenuation in order to highlight anisotropic scattering. Pixel-by-pixel comparison of LD images with LR and multiphoton images demonstrates that LD is produced by under-resolved structures that are not revealed by other means, notably within the sarcomere of skeletal muscles. LD microscopy appears as a powerful tool to provide new insights into the macro-molecular organization of biological specimens at the sub-microscopic scale without labelling.

Optimization of fast spectrally encoded Mueller polarimeters for real-time monitoring.

In this paper, we report a generalized theoretical framework on spectrally encoded polarimeters to display in real time both linear and circular retardance as well as linear and circular diattenuation on the basis of a recent experimental work detailed in Sci. Rep.9, 3972 (2019)SRCEC32045-232210.1038/s41598-019-40467-z. The considered polarimeters use a broadband light source and wavelength-dependent retarders whose thicknesses are selected to guarantee minimal noise propagation and reduced data processing for real-time display of pure polarimetric effects.

Fast spectrally encoded Mueller optical scanning microscopy.

Mueller microscopes enable imaging of the optical anisotropic properties of biological or non-biological samples, in phase and amplitude, at sub-micrometre scale. However, the development of Mueller microscopes poses an instrumental challenge: the production of polarimetric parameters must be sufficiently quick to ensure fast imaging, so that the evolution of these parameters can be visualised in real-time, allowing the operator to adjust the microscope while constantly monitoring them. In this report, a full Mueller scanning microscope based on spectral encoding of polarization is presented. The spectrum, collected every 10 µs for each position of the optical beam on the specimen, incorporates all the information needed to produce the full Mueller matrix, which allows simultaneous display of all the polarimetric parameters, at the unequalled rate of 1.5 Hz (for an image of 256 × 256 pixels). The design of the optical blocks allows for the real-time display of linear birefringent images which serve as guidance for the operator. In addition, the instrument has the capability to easily switch its functionality from a Mueller to a Second Harmonic Generation (SHG) microscope, providing a pixel-to-pixel matching of the images produced by the two modalities. The device performance is illustrated by imaging various unstained biological specimens.

Recovering distance information in spectral domain interferometry.

This work evaluates the performance of the Complex Master Slave (CMS) method, that processes the spectra at the interferometer output of a spectral domain interferometry device without involving Fourier transforms (FT) after data acquisition. Reliability and performance of CMS are compared side by side with the conventional method based on FT, phase calibration with dispersion compensation (PCDC). We demonstrate that both methods provide similar results in terms of resolution and sensitivity drop-off. The mathematical operations required to produce CMS results are highly parallelizable, allowing real-time, simultaneous delivery of data from several points of different optical path differences in the interferometer, not possible via PCDC.

Group refractive index and group velocity dispersion measurement by complex master slave interferometry.

This paper demonstrates that the complex master slave interferometry (CMSI) method used in spectral domain interferometry (SDI) can efficiently be used for accurate refractive index and group velocity dispersion measurements of optically transparent samples. For the first time, we demonstrate the relevance of the phase information delivered by CMSI for dispersion evaluations with no need to linearize data. The technique proposed here has been used to accurately measure the group refractive index and the group velocity dispersion of a strong dispersive sample (SF6 glass), and a weak dispersive one (distilled water). The robustness of the technique is demonstrated through the manipulation of several sets of experimental data.

Polarization-resolved second harmonic microscopy of skeletal muscle in sepsis.

Polarization-resolved second harmonic generation (P-SHG) microscopy is able to probe the sub-micrometer structural organization of myosin filaments within skeletal muscle. In this study, P-SHG microscopy was used to analyze the structural consequences of sepsis, which is the main cause of the critical illness polyneuromyopathy (CIPNM). Experiments conducted on two populations of rats demonstrated a significant difference of the anisotropy parameter between healthy and septic groups, indicating that P-SHG microscopy is promising for the diagnosis of CIPNM. The difference, which can be attributed to a change of myosin conformation at the sub-sarcomere scale, cannot be evidenced by classical SHG imaging.

Snapshot second-harmonic generation polarimeter based on spectral analysis.

A new high-speed second-harmonic generation (SHG) polarimetric method is reported. It is based on the spectral analysis of the SHG radiation emitted by a nonlinear medium excited with circularly polarized femtosecond laser pulses. The setup uses only passive components for polarization encoding and a fast spectrometer for spectral analysis. The method is validated on a z-cut quartz plate, then on a collagen-rich biological tissue with a view to single image polarization-resolved SHG microscopy.

Passive optical module for polarization-sensitive optical coherence tomography systems.

The paper presents a proof-of-concept polarization-sensitive swept source optical coherence tomography (OCT) system that performs measurements of the retardance as well as of the axis orientation of a linear birefringent sample. The system performs single input state polarization-sensitive OCT and employs an optical module based on optically passive elements such as two beam displacers and a Faraday rotator. Our implementation of the PS-OCT system does not need any calibration step to compensate for the polarimetric effect of the fibers, and its operation does not require a balanced polarization-diversity detector. The optical module allows measurement of the two polarization properties of the sample via two measurements which are performed simultaneously.

Gabor fusion master slave optical coherence tomography.

This paper describes the application of the Gabor filtering protocol to a Master/Slave (MS) swept source optical coherence tomography (SS)-OCT system at 1300 nm. The MS-OCT system delivers information from selected depths, a property that allows operation similar to that of a time domain OCT system, where dynamic focusing is possible. The Gabor filtering processing following collection of multiple data from different focus positions is different from that utilized by a conventional swept source OCT system using a Fast Fourier transform (FFT) to produce an A-scan. Instead of selecting the bright parts of A-scans for each focus position, to be placed in a final B-scan image (or in a final volume), and discarding the rest, the MS principle can be employed to advantageously deliver signal from the depths within each focus range only. The MS procedure is illustrated on creating volumes of data of constant transversal resolution from a cucumber and from an insect by repeating data acquisition for 4 different focus positions. In addition, advantage is taken from the tolerance to dispersion of the MS principle that allows automatic compensation for dispersion created by layers above the object of interest. By combining the two techniques, Gabor filtering and Master/Slave, a powerful imaging instrument is demonstrated. The master/slave technique allows simultaneous display of three categories of images in one frame: multiple depth en-face OCT images, two cross-sectional OCT images and a confocal like image obtained by averaging the en-face ones. We also demonstrate the superiority of MS-OCT over its FFT based counterpart when used with a Gabor filtering OCT instrument in terms of the speed of assembling the fused volume. For our case, we show that when more than 4 focus positions are required to produce the final volume, MS is faster than the conventional FFT based procedure.

Scanning Mueller polarimetric microscopy.

A full Mueller polarimeter was implemented on a commercial laser-scanning microscope. The new polarimetric microscope is based on high-speed polarization modulation by spectral coding using a wavelength-swept laser as a source. Calibration as well as estimation of the measurement errors of the device are reported. The acquisition of Mueller images at the speed of a scanning microscope is demonstrated for the first time. Mueller images of mineral and biological samples illustrate this new polarimetric microscopy.

Master/slave interferometry - ideal tool for coherence revival swept source optical coherence tomography.

In this paper, we demonstrate that the master slave (MS) interferometry method can significantly simplify the practice of coherence revival swept source optical coherence tomography (OCT) technique. Previous implementations of the coherence revival technique required considerable resources on dispersion compensation and data resampling. The total tolerance of the MS method to nonlinear tuning, to dispersion in the interferometer and to dispersion due to the laser cavity, makes the MS ideally suited to the practice of coherence revival. In addition, enhanced versatility is allowed by the MS method in displaying shorter axial range images than that determined by the digital sampling of the data. This brings an immediate improvement in the speed of displaying cross-sectional images at high rates without the need of extra hardware such as graphics processing units or field programmable gate arrays. The long axial range of the coherence revival regime is proven with images of the anterior segment of healthy human volunteers.

Complex master slave interferometry.

A general theoretical model is developed to improve the novel Spectral Domain Interferometry method denoted as Master/Slave (MS) Interferometry. In this model, two functions, g and h are introduced to describe the modulation chirp of the channeled spectrum signal due to nonlinearities in the decoding process from wavenumber to time and due to dispersion in the interferometer. The utilization of these two functions brings two major improvements to previous implementations of the MS method. A first improvement consists in reducing the number of channeled spectra necessary to be collected at Master stage. In previous MSI implementation, the number of channeled spectra at the Master stage equated the number of depths where information was selected from at the Slave stage. The paper demonstrates that two experimental channeled spectra only acquired at Master stage suffice to produce A-scans from any number of resolved depths at the Slave stage. A second improvement is the utilization of complex signal processing. Previous MSI implementations discarded the phase. Complex processing of the electrical signal determined by the channeled spectrum allows phase processing that opens several novel avenues. A first consequence of such signal processing is reduction in the random component of the phase without affecting the axial resolution. In previous MSI implementations, phase instabilities were reduced by an average over the wavenumber that led to reduction in the axial resolution.

70 kHz full 4x4 Mueller polarimeter and simultaneous fiber calibration for endoscopic applications.

A new set-up is proposed to measure the full polarimetric properties of a sample through an optical fiber, paving the way to full-Mueller endoscopic imaging. The technique combines a channeled spectrum polarimeter and an interferometer. This permits high-speed measurement of two Mueller matrices simultaneously. The first matrix characterizes only the fiber while the second characterizes both fiber and sample. The instrument is validated on vacuum, a quarter-wave plate and a linear polarizer for single-point measurements. Insensitivity of the polarimetric measurement to fiber disturbances is proven while manipulating the fiber.

Polarization-sensitive optical coherence tomography system tolerant to fiber disturbances using a line camera.

This Letter presents a spectral-domain, polarization-sensitive optical coherence tomography (PS-OCT) system, where the light collection from the two arms of the interferometer is performed exclusively using single-mode fibers and couplers, and the two orthogonal polarization components are sequentially detected by a single line camera. Retardance measurements can be affected by polarimetric effects because of fiber birefringence and diattenuation in fiber couplers. This configuration bypasses such issues by performing polarization selection before the collection fiber through the combination of a polarization rotator and a linear polarizer. Retardance calibration is achieved with a Berek compensator. Similar net retardance maps of a birefringent phantom are obtained for two different settings of induced fiber birefringence, effectively demonstrating the tolerance of the configuration to fiber-based disturbances.

Analysis of experimental depolarizing Mueller matrices through a hybrid decomposition.

We propose a new decomposition for depolarizing Mueller matrices. This decomposition, which consists of a product of four basic optical devices (two diattenuators, a retarder, and a depolarizer) is derived from a previous one known as "symmetric decomposition" [J. Opt. Soc. Am. A26, 1109 (2009)10.1364/JOSAA.26.001109JOAOD61084-7529] and makes it easier to interpret polarization properties of Mueller matrices and improves estimation of the extracted parameters. Its application is illustrated by several theoretical and experimental examples.

100 kHz Mueller polarimeter in reflection configuration.

A new setup is proposed to perform high-speed Mueller polarimetry by spectral coding of polarization in a reflection configuration. The system uses a swept laser source and a photodiode, which results in a simple optical setup that allows measurement of Mueller matrices at 100 kHz repetition rate. A special focus is made on the influence of the cube beam splitter polarimetric response, which is essential to measurements in a reflection configuration. The instrument is first validated on reference samples for single-point measurements, and the effect of a proper system calibration is also demonstrated on polarimetric images. The device is intended to be implemented within a laser scanning microscope to perform multimodal imaging (confocal/multiphoton and Mueller polarimetry).