Quantification of collagen fiber properties in alcoholic liver fibrosis using polarization-resolved second harmonic generation microscopy.

Liver fibrosis is assessed mainly by conventional staining or second harmonic generation (SHG) microscopy, which can only provide collagen content in fibrotic area. We propose to use polarization-resolved SHG (PR-SHG) microscopy to quantify liver fibrosis in terms of collagen fiber orientation and crystallization. Liver samples obtained from autopsy cases with fibrosis stage of F0-F4 were evaluated with an SHG microscope, and 12 consecutive PR-SHG images were acquired while changing the polarization azimuth angle of the irradiated laser from 0° to 165° in 15° increments using polarizer. The fiber orientation angle (φ) and degree (ρ) of collagen were estimated from the images. The SHG-positive area increased as the fibrosis stage progressed, which was well consistent with Sirius Red staining. The value of φ was random regardless of fibrosis stage. The mean value of ρ (ρ-mean), which represents collagen fiber crystallinity, varied more as fibrosis progressed to stage F3, and converged to a significantly higher value in F4 than in other stages. Spatial dispersion of ρ (ρ-entropy) also showed increased variation in the stage F3 and decreased variation in the stage F4. It was shown that PR-SHG could provide new information on the properties of collagen fibers in human liver fibrosis.

Carrier conversion from terahertz wave to dual-wavelength near-infrared light for photonic terahertz detection in wireless communication.

THz waves are promising wireless carriers for next-generation wireless communications, where a seamless connection from wireless to optical communication is required. In this study, we demonstrate carrier conversion from THz waves to dual-wavelength NIR light injection-locking to an optical frequency comb using asynchronous nonpolarimetric electro-optic downconversion with an electro-optic polymer modulator. THz wave in the W band was detected as a stable photonic RF beat signal of 1 GHz with a signal-to-noise ratio of 20 dB via the proposed THz-to-NIR carrier conversion. In addition, the results imply the potential of the photonic detection of THz waves for wireless-to-optical seamless communication.

Real-time hybrid angular-interrogation surface plasmon resonance sensor in the near-infrared region for wide dynamic range refractive index sensing.

Herein, we integrated angle-scanning surface plasmon resonance (SPR) and angle-fixed SPR as a hybrid angular-interrogation SPR to enhance the sensing performance. Galvanometer-mirror-based beam angle scanning achieves a 100-Hz acquisition rate of both the angular SPR reflectance spectrum and the angle-fixed SPR reflectance, whereas the use of near-infrared light enhances the refractive index (RI) sensitivity, range, and precision compared with visible light. Simultaneous measurement of the angular SPR reflectance spectrum and angle-fixed SPR reflectance boosts the RI change range, RI resolution, and RI accuracy to 10-1-10-6 RIU, 2.24 × 10-6 RIU, and 5.22 × 10-6 RIU, respectively. The proposed hybrid SPR is a powerful tool for wide-dynamic-range RI sensing with various applications.

Nanometer-precision surface metrology of millimeter-sized stepped objects using full-cascade-linked synthetic-wavelength digital holography using a line-by-line full-mode-extracted optical frequency comb.

Digital holography (DH) is a powerful tool for the surface profilometry of objects with sub-wavelength precision. In this article, we demonstrate full-cascade-linked synthetic-wavelength DH for nanometer-precision surface metrology of millimeter-sized stepped objects. 300 modes of optical frequency comb (OFC) with different wavelengths are sequentially extracted at a step of mode spacing from a 10GHz-spacing, 3.72THz-spanning electro-optic modulator OFC. The resulting 299 synthetic wavelengths and a single optical wavelength are used to generate a fine-step wide-range cascade link covering within a wavelength range of 1.54 µm to 29.7 mm. We determine the sub-millimeter and millimeter step differences with axial uncertainty of 6.1 nm within the maximum axial range of 14.85 mm.

Assessment of Ultra-Early-Stage Liver Fibrosis in Human Non-Alcoholic Fatty Liver Disease by Second-Harmonic Generation Microscopy.

Non-alcoholic fatty liver disease (NAFLD) is associated with the chronic progression of fibrosis. In general, the progression of liver fibrosis is determined by a histopathological assessment with a collagen-stained section; however, the ultra-early stage of liver fibrosis is challenging to identify because of the low sensitivity in the collagen-selective staining method. In the present study, we demonstrate the feasibility of second-harmonic generation (SHG) microscopy in the histopathological diagnosis of the liver of NAFLD patients for the quantitative assessment of the ultra-early stage of fibrosis. We investigated four representative NAFLD patients with early stages of fibrosis. SHG microscopy visualised well-matured fibrotic structures and early fibrosis diffusely involving liver tissues, whereas early fibrosis is challenging to be identified by conventional histopathological methods. Furthermore, the SHG emission directionality analysis revealed the maturation of each collagen fibre of each patient. As a result, SHG microscopy is feasible for assessing liver fibrosis on NAFLD patients, including the ultra-early stage of liver fibrosis that is difficult to diagnose by the conventional histopathological method. The assessment method of the ultra-early fibrosis by using SHG microscopy may serve as a crucial means for pathological, clinical, and prognostic diagnosis of NAFLD patients.

Ultralow-frequency ultranarrow-bandwidth coherent terahertz imaging for nondestructive testing of mortar material.

Nondestructive testing of concrete materials is essential in civil engineering to maintain social infrastructure such as buildings or bridges. In this study, we constructed an ultralow-frequency, ultranarrow-bandwidth, coherent terahertz (THz) imaging system based on THz time-domain spectroscopy (THz-TDS). Based on its ultralow-frequency-localized THz wave and coherent detection, the present system achieved a wide dynamic range of THz power over 100 dB at 0.046 THz, which is appropriate to measure the mortar material. The achieved dynamic range of the THz power was 59 dB larger than that of a commercialized THz-TDS system and 49 dB larger than that of an ultralow-frequency noncoherent THz imaging system equipped with a high-power electric THz source. Ultimately, the proposed system could visualize the inner structure of a mortar sample with a thickness of 10 mm, and the present system can investigate a mortar sample with a thickness of over 130 mm. The proposed method is an attractive tool for non-destructive testing of thick concrete structures characterized by non-invasiveness and non-contact remoteness.

Synthesis of D-π-A type benzothiazole-pyridinium salt composite and its application as photo-degradation agent for amyloid fibrils.

We have synthesized a cyan fluorescent benzothiazole-pyridinium salt composite based on D-π-A architecture. This salt was found to work as not only a two- and three-photon excitable fluorophore but also a degradation agent against amyloid fibrils under LED irradiation conditions.

Multicascade-linked synthetic-wavelength digital holography using a line-by-line spectral-shaped optical frequency comb.

Phase imaging without a phase wrapping ambiguity is required for wide-axial-range 3D imaging in the fields of surface topography measurement and biomedical imaging. Although multicascade-linked synthetic-wavelength digital holography (MCL-SW-DH) using an optical frequency synthesizer (OFS) is a promising method to meet this requirement, the slow switching of multiple optical wavelengths in the OFS prevents rapid imaging. In the work described in this article, a line-by-line spectral-shaped electro-optics-modulator-based optical frequency comb (EOM-OFC) is used as a light source in MCL-SW-DH to achieve rapid image acquisition. While MCL-SW-DH enables surface topography measurement with millimeter-order axial range and micrometer-order axial resolution, the line-by-line spectral-shaped EOM-OFC extracts a single narrow-linewidth OFC mode from the 10 GHz-spacing EOM-OFC at a center wavelength of 1545 nm within a spectral range of 30 nm at an interval of 500 ms. The effectiveness of the proposed MCL-SW-DH was highlighted by performing surface topography measurement with four step differences of sub-millimeter to millimeter size with an axial uncertainty of 2.08 µm in the image acquisition time of several seconds. The proposed MCL-SW-DH will be a powerful tool for 3D imaging with a wide axial range and high axial resolution.

Two-photon excitable boron complex based on tridentate imidazo[1,5-a]pyridine ligand for heavy-atom-free mitochondria-targeted photodynamic therapy.

We have synthesized a cyan fluorescent boron complex based on a tridentate imidazo[1,5-a]pyridine ligand. The boron complex was found to have potential applications as not only a chiroptical material but also a heavy-atom-free mitochondria-targeted photosensitizer for cancer treatment.

Molecular imaging analysis of microvesicular and macrovesicular lipid droplets in non-alcoholic fatty liver disease by Raman microscopy.

Predominant evidence of non-alcoholic fatty liver disease (NAFLD) is the accumulation of excess lipids in the liver. A small group with NAFLD may have a more serious condition named non-alcoholic steatohepatitis (NASH). However, there is a lack of investigation of the accumulated lipids with spatial and molecular information. Raman microscopy has the potential to characterise molecular species and structures of lipids based on molecular vibration and can achieve high spatial resolution at the organelle level. In this study, we aim to demonstrate the feasibility of Raman microscopy for the investigation of NAFLD based on the molecular features of accumulated lipids. By applying the Raman microscopy to the liver of the NASH model mice, we succeeded in visualising the distribution of lipid droplets (LDs) in hepatocytes. The detailed analysis of Raman spectra revealed the difference of molecular structural features of the LDs, such as the degree of saturation of lipids in the LDs. We also found that the inhomogeneous distribution of cholesterol in the LDs depending on the histology of lipid accumulation. We visualised and characterised the lipids of NASH model mice by Raman microscopy at organelle level. Our findings demonstrated that the Raman imaging analysis was feasible to characterise the NAFLD in terms of the molecular species and structures of lipids.

Two- and three-photon excitable quaternized imidazo[1,2-a]pyridines as mitochondrial imaging and potent cancer therapy agents.

We have synthesized a series of quaternized imidazo[1,2-a]pyridines in three steps from commercially available reagents. These compounds exhibit blue fluorescence emission at around 425 nm with good quantum yields. In addition, one specific compound was found to work as not only a two- and three-photon excitable mitochondria imaging agent, but also a therapeutic agent upon continuous irradiation conditions.

Dynamic characterization of polarization property in liquid-crystal-on-silicon spatial light modulator using dual-comb spectroscopic polarimetry.

Spectroscopic polarimetry (SP) is a powerful tool for characterization of thin film, polarization optics, semiconductor, and others. However, mechanical polarization modulation of broadband light hampers its application for dynamic monitoring of a sample. In this article, we demonstrate the dynamic SP with features of polarization-modulation-free polarimetry and spectrometer-free spectroscopy benefiting from dual-comb spectroscopy (DCS) using a pair of optical frequency combs (OFCs). DCS enables the direct determination of polarization without the need for polarization modulation by using mode-resolved OFC spectra of amplitude and phase for two orthogonally linear-polarized lights while securing rapid, high-precision, broadband spectroscopy without the need for spectrometer. Effectiveness of the proposed system is highlighted by visualizing the hysteresis property of dynamic response in a liquid-crystal-on-silicon spatial light modulator at a sampling rate of 105 Hz.

Optical image amplification in dual-comb microscopy.

Dual-comb microscopy (DCM), based on a combination of dual-comb spectroscopy (DCS) with two-dimensional spectral encoding (2D-SE), is a promising method for scan-less confocal laser microscopy giving an amplitude and phase image contrast with the confocality. However, signal loss in a 2D-SE optical system hampers increase in image acquisition rate due to decreased signal-to-noise ratio. In this article, we demonstrated optical image amplification in DCM with an erbium-doped fiber amplifier (EDFA). Combined use of the image-encoded DCS interferogram and the EDFA benefits from not only the batch amplification of amplitude and phase images but also significant rejection of amplified spontaneous emission (ASE) background. Effectiveness of the optical-image-amplified DCM is highlighted in the single-shot quantitative nanometer-order surface topography and the real-time movie of polystyrene beads dynamics under water convection. The proposed method will be a powerful tool for real-time observation of surface topography and fast dynamic phenomena.

One-Drop Self-Assembly of Ultra-Fine Second-Order Organic Nonlinear Optical Crystal Nanowires.

In this study, we propose a one-drop self-assembly method, which proved capable of successfully preparing 4-N, N-dimethylamino-4'-N'-methyl-stilbazolium tosylate (DAST) single-crystalline nanowires (NWs). The apparent roughness of the DAST NWs was determined to be less than 100 pm by using a high-resolution atomic force microscope, indicating their ultrafine quality. The DAST NWs also exhibited excellent nonlinear optical properties, including two-photon excited fluorescence and second harmonic generation, which could enable the production of low-cost, low-power-consumption wideband wavelength conversion devices. Thus, the described method may provide a new avenue for organic NW fabrication.

Quantitative in situ time-series evaluation of osteoblastic collagen synthesis under cyclic strain using second-harmonic-generation microscopy.

The aim of this study is to evaluate the osteoblastic collagen synthesis under mechanical stimulation using second-harmonic-generation (SHG) microscopy. We apply SHG microscopy to monitor the collagen fibers synthesized by osteoblast-like cells (MC3T3-E1) without the need for fixation and staining. To quantitatively evaluate the influence of mechanical stimulation on osteoblastic collagen synthesis, we compare SHG images of osteoblast-synthesized collagen fibers with and without a cyclic stretch stimulus applied using a lab-made stretching device. We acquire SHG images every 7 days for 3 weeks at different stimulus conditions (5 min/day and 3 h/day with a strain magnitude of 5% and a frequency of 0.5 Hz). Image analysis of the average SHG intensity indicates that the amount of osteoblastic collagen synthesis is significantly enhanced by the cyclic stretch compared with the nonstretched condition, while there is no significant difference between the two mechanical stimulation conditions. Furthermore, the maturity of the collagen fibers in the early stage of bone formation is not affected by the mechanical stimulation. The results can be used in bone regenerative medicine to apply feedback control of collagen synthesis by artificial stimulation.

Texture analysis of second-harmonic-generation images for quantitative analysis of reticular dermal collagen fibre in vivo in human facial cheek skin.

Second-harmonic-generation (SHG) microscopy is a powerful tool for in vivo visualisation of collagen fibres in human skin because of its specific collagen selectivity without the need for staining, non-invasiveness and high-resolution three-dimensional imaging. Although texture analysis of SHG images is a promising method for the quantitative analysis of well-orientated collagen fibre structure in the tendon and cornea, there are few attempts to assess cutaneous ageing. In this study, we applied two texture analysis techniques, namely autocorrelation (2D-AC) analysis and two-dimensional Fourier transform (2D-FT), to evaluate the age-dependent changes in reticular dermal collagen fibres in in vivo human cheek skin. Age-dependent changes in the reticular dermal collagen fibres of female subjects in their 20s, 40s and 60s clearly appeared in these texture analyses. Furthermore, the parameter from 2D-AC analysis showed a significantly higher correlation with skin elasticity measured by a Cutometer® . These results clearly indicate that 2D-AC analysis of SHG images is highly promising for the quantitative evaluation of age-dependent change in facial collagen fibres as well as skin elasticity. An appropriate texture analysis will help to provide quantitative insight into collagen fibre structure and will be useful for the diagnosis of pathological conditions as well as cutaneous ageing in skin.

Strain sensing based on strain to radio-frequency conversion of optical frequency comb.

We propose an optical frequency comb (OFC)-based strain sensing method, namely OFC sensing cavity, which is capable of radio-frequency (RF)-based strain measurement. We developed a null-method-based strain sensing system with a comb-spacing-stabilized OFC generator. We realized strain measurement from 1.83 µÎµ to 1800 µÎµ with a sensing fiber length of 20 mm. The measurable strain frequency range of the developed strain sensing system was from 0 to 310 Hz. Owing to the use of RF-based strain measurement, our approach would be a useful and powerful tool for sensing of strain or other physical quantities, and the concept of the OFC sensing cavity is a new aspect of OFC technology.

Dual-comb spectroscopic ellipsometry.

Spectroscopic ellipsometry is a means of investigating optical and dielectric material responses. Conventional spectroscopic ellipsometry is subject to trade-offs between spectral accuracy, resolution, and measurement time. Polarization modulation has afforded poor performance because of its sensitivity to mechanical vibrational noise, thermal instability, and polarization-wavelength dependency. We combine spectroscopic ellipsometry with dual-comb spectroscopy, namely, dual-comb spectroscopic ellipsometry. Dual-comb spectroscopic ellipsometry (DCSE). DCSE directly and simultaneously obtains the ellipsometric parameters of the amplitude ratio and phase difference between s-polarized and p-polarized light signals with ultra-high spectral resolution and no polarization modulation, beyond the conventional limit. Ellipsometric evaluation without polarization modulation also enhances the stability and robustness of the system. In this study, we construct a polarization-modulation-free DCSE system with a spectral resolution of up to 1.2 × 10-5 nm throughout the spectral range of 1514-1595 nm and achieved an accuracy of 38.4 nm and a precision of 3.3 nm in the measurement of thin-film samples.Spectroscopic ellipsometry is an established technique to characterize the optical properties of a material. Here, Minamikawa et al. combine the method with dual-comb spectroscopy, which allows them to obtain ellipsometric parameters including the phase difference between s-polarized and p-polarized light.

In situ time-series monitoring of collagen fibers produced by standing-cultured osteoblasts using a second-harmonic-generation microscope.

In bone tissue engineering and regeneration, there is a considerable need for an unstained method of monitoring collagen fibers produced by osteoblasts. This is because collagen fibers play an important role as a bone matrix and continuous monitoring of their temporal dynamics is important in clarifying the organization process toward forming bone tissue. In the work described here, using a second-harmonic-generation (SHG) microscope, we performed in situ time-series monitoring of collagen fibers produced by cultured osteoblasts without the need for staining. Use of the 19 fs near-infrared pulsed light enables us to visualize the temporal dynamics in a thin layer of collagen fibers produced by a single layer of osteoblasts in high-contrast SHG images. While the collagen fibers were produced and stored inside the osteoblasts at an early stage of culturing, the network structure of collagen fibers was formed and locally condensed at a late stage. Furthermore, we extracted a quantitative parameter of collagen maturity degree in the cultured sample by use of image analysis based on a two-dimensional Fourier transform of the SHG image. The proposed method will be useful for in situ quality and quantity control of collagen fibers in bone tissue engineering and regeneration.

Motion-artifact-robust, polarization-resolved second-harmonic-generation microscopy based on rapid polarization switching with electro-optic Pockells cell and its application to in vivo visualization of collagen fiber orientation in human facial skin.

Polarization-resolved second-harmonic-generation (PR-SHG) microscopy is a powerful tool for investigating collagen fiber orientation quantitatively with low invasiveness. However, the waiting time for the mechanical polarization rotation makes it too sensitive to motion artifacts and hence has hampered its use in various applications in vivo. In the work described in this article, we constructed a motion-artifact-robust, PR-SHG microscope based on rapid polarization switching at every pixel with an electro-optic Pockells cell (PC) in synchronization with step-wise raster scanning of the focus spot and alternate data acquisition of a vertical-polarization-resolved SHG signal and a horizontal-polarization-resolved one. The constructed PC-based PR-SHG microscope enabled us to visualize orientation mapping of dermal collagen fiber in human facial skin in vivo without the influence of motion artifacts. Furthermore, it implied the location and/or age dependence of the collagen fiber orientation in human facial skin. The robustness to motion artifacts in the collagen orientation measurement will expand the application scope of SHG microscopy in dermatology and collagen-related fields.