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.

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.

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.

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.

A non-hypocholesterolemic atorvastatin treatment improves vessel elasticity by acting on elastin composition in WHHL rabbits.

BACKGROUND AND AIMS: Statins are prescribed for their preventative effects within atherosclerosis development. To our knowledge, no study focusing on very low-dose (non-hypolipidemic effect) and long-term atorvastatin treatment in vivo was available. Our aim was to assess the effect of such atorvastatin treatment on the mechanical and functional characteristics of arteries in the context of primary prevention. METHODS: An atorvastatin treatment (2.5 mg/kg/day) was tested against controls on 34 male 3 to 12 month-old WHHL rabbits. No effect on total cholesterol, triglycerides, HDL or LDL was observed. The arterial stiffness was evaluated on vigil animals by pulse wave velocity (PWV) measurement. Then, in vitro measurements were made to evaluate (1) the endothelial and vascular smooth muscle function, (2) the elasticity of the arterial wall and (3) the composition in collagen and elastin in the aorta. RESULTS: The PWV increasing observed with age in control group was canceled by treatment, creating a significance difference between groups at 12 months (5.17 ± 0.50 vs 2.14 ± 0.34 m s(-1) in control and treated groups respectively). Vasoreactivity modifications can't explain this result but maintain of elasticity with treatment in large arteries was confirm by a static tensile test. A first possible explanation is the change of wall composition with treatment, validated by the percentage of elastin at 12 months, 4.4% lower in the control group compared to the treated group (p < 0.05). CONCLUSIONS: This study shows that a non-hypocholesterolemic statin treatment could improve vessel elasticity in the atherosclerotic WHHL model. The great novelty of this work is the vessel wall composition changing associated. This first approach in animal opens the reflection on the use of these low doses in humans. This could be interesting in the context of arterial stiffening with aging, non-hyperlipidemic atherosclerosis or with cholesterol reduce by another therapy or lifestyle modification.

Evaluation of area-based collagen scoring by nonlinear microscopy in chronic hepatitis C-induced liver fibrosis.

In this paper we analyze a fibrosis scoring method based on measurement of the fibrillar collagen area from second harmonic generation (SHG) microscopy images of unstained histological slices from human liver biopsies. The study is conducted on a cohort of one hundred chronic hepatitis C patients with intermediate to strong Metavir and Ishak stages of liver fibrosis. We highlight a key parameter of our scoring method to discriminate between high and low fibrosis stages. Moreover, according to the intensity histograms of the SHG images and simple mathematical arguments, we show that our area-based method is equivalent to an intensity-based method, despite saturation of the images. Finally we propose an improvement of our scoring method using very simple image processing tools.

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).

Measuring the scattering coefficient of turbid media from two-photon microscopy.

In this paper, we propose a new and simple method based on two-photon excitation fluorescence (TPEF) microscopy to measure the scattering coefficient µ(s) of thick turbid media. We show, from Monte Carlo simulations, that µ(s) can be derived from the axial profile of the ratio of the TPEF signals epi-collected by the confocal and the non-descanned ports of a scanning microscope, independently of the anisotropy factor g and of the absorption coefficient µ(a) of the medium. The method is validated experimentally on tissue-mimicking optical phantoms, and is shown to have potential for imaging the scattering coefficient of heterogeneous media.

Two-photon microscopy of dermal innervation in a human re-innervated model of skin.

When skin is injured, innervation can be severely disrupted. The subsequent re-innervation processes are poorly understood notably because of the inability to image the full meandering course of nerves with their ramifications and endings from histological slices. In this letter, we report on two-photon excitation fluorescence (TPEF) microscopy of entire human skin explants re-innervated by rodent sensory neurons labelled with the styryl dye FM1-43. TPEF imaging of nerve fibres to a depth up to roughly 300 µm within the dermis was demonstrated, allowing three-dimensional reconstruction of the neural tree structure. Endogenous second-harmonic imaging of type I fibrillar collagen was performed in parallel to TPEF imaging using the same nonlinear microscope, revealing the path of the nerves through the dermis.

Mueller matrix polarimetry for improved liver fibrosis diagnosis.

An experimental Mueller matrix polarimeter is used to quantify human liver fibrosis by measuring retardance and depolarization of thin biopsies. The former parameter is sensitive to fibrillar collagen, the latter is specifically sensitive to fibrillar collagen around blood vessels, which is not significant for liver fibrosis diagnosis. By using depolarization like a filter, retardance distribution enables distinguishing between disease stages and limits the high degree of observer discrepancy.

A robust collagen scoring method for human liver fibrosis by second harmonic microscopy.

Second Harmonic Generation (SHG) microscopy offers the opportunity to image collagen of type I without staining. We recently showed that a simple scoring method, based on SHG images of histological human liver biopsies, correlates well with the Metavir assessment of fibrosis level (Gailhouste et al., J. Hepatol., 2010). In this article, we present a detailed study of this new scoring method with two different objective lenses. By using measurements of the objectives point spread functions and of the photomultiplier gain, and a simple model of the SHG intensity, we show that our scoring method, applied to human liver biopsies, is robust to the objective's numerical aperture (NA) for low NA, the choice of the reference sample and laser power, and the spatial sampling rate. The simplicity and robustness of our collagen scoring method may open new opportunities in the quantification of collagen content in different organs, which is of main importance in providing diagnostic information and evaluation of therapeutic efficiency.

Fibrillar collagen scoring by second harmonic microscopy: a new tool in the assessment of liver fibrosis.

BACKGROUND & AIMS: Imaging of supramolecular structures by multiphoton microscopy offers significant advantages for studying specific fibrillar compounds in biological tissues. In this study, we aimed to demonstrate the relevance of Second Harmonic Generation (SHG) for assessing and quantifying, without staining, fibrillar collagen in liver fibrosis. METHODS: We first showed the relationship between SHG signal and collagen forms over-produced and accumulated during fibrosis progression. Taking this property into consideration, we developed an innovative method to precisely quantify the fibrosis area in histological slices by scoring of fibrillar collagen deposits (Fibrosis-SHG index). RESULTS: The scoring method was routinely applied to 119 biopsies from patients with chronic liver disease allowing a fast and accurate measurement of fibrosis correlated with the Fibrosis-Metavir score (rho=0.75, p<0.0001). The technique allowed discriminating patients with advanced (moderate to severe) fibrosis (AUROC=0.88, p<0.0001) and cirrhosis (AUROC=0.89, p<0.0001). Taking advantage of its continuous gradation, the Fibrosis-SHG index also allowed the discrimination of several levels of fibrosis within the same F-Metavir stage. The SHG process presented several advantages such as a high reliability and sensitivity that lead to a standardized evaluation of hepatic fibrosis in liver biopsies without staining and pathological examination. CONCLUSIONS: Second harmonic microscopy emerges as an original and powerful tool in the assessment of liver fibrosis and offers new possibilities for the evaluation of experimental protocols. We expect that this technology could easily be applicable in the study of other fibro-proliferative pathologies.

Second harmonic microscopy of axonemes.

We performed Second Harmonic Microscopy of axonemes obtained from sea urchin sperm. Using polarization analysis and a trade-off between signal and photodamage, we were able to determine, for the first time to our knowledge, the nonlinear susceptibility chizxx/chixzx = 1.1+/-0.2 and chizzz/chixzx = 4+/-0.5 of axonemes.

Collagen and myosin characterization by orientation field second harmonic microscopy.

Collagen and myosin fibrils are endogenous harmonophores that both give rise to Second Harmonic Generation (SHG). By combining four polarization SHG images provided by a scanning microscope, we show that the orientation of the principal axis of the nonlinear susceptibility tensor chi(2) can be determined for each pixel of the image. The ratio rho = chi33/chi15 of the principal components of chi(2) of collagen and myosin was obtained with the same method, and found within the range 1.6-1.8 and 0.5-0.6 respectively. The orientation of the principal axis of chi(2) is shown to be correlated to the orientation of the fibrils themselves. This provides a straightforward method, which we call Orientation Field-Second Harmonic Microscopy (OF-SHM), to reconstruct orientation fields of fibrils at various scales and resolutions in different biological systems (from muscle sarcomere to the whole embryo).