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.

Rapid, high-sensitivity detection of biomolecules using dual-comb biosensing.

Rapid, sensitive detection of biomolecules is important for biosensing of infectious pathogens as well as biomarkers and pollutants. For example, biosensing of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is still strongly required for the fight against coronavirus disease 2019 (COVID-19) pandemic. Here, we aim to achieve the rapid and sensitive detection of SARS-CoV-2 nucleocapsid protein antigen by enhancing the performance of optical biosensing based on optical frequency combs (OFC). The virus-concentration-dependent optical spectrum shift produced by antigen-antibody interactions is transformed into a photonic radio-frequency (RF) shift by a frequency conversion between the optical and RF regions in the OFC, facilitating rapid and sensitive detection with well-established electrical frequency measurements. Furthermore, active-dummy temperature-drift compensation with a dual-comb configuration enables the very small change in the virus-concentration-dependent signal to be extracted from the large, variable background signal caused by temperature disturbance. The achieved performance of dual-comb biosensing will greatly enhance the applicability of biosensors to viruses, biomarkers, environmental hormones, and so on.

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.

Definitive Confirmation of Erythropoietic Protoporphyria via Re-biopsy Three Years After Initial Liver Biopsy at Age 15.

Erythropoietic protoporphyria (EPP) is a rare inherited disorder of porphyrin metabolism that can cause liver damage and cholestatic hepatocellular failure. We report a case of EPP in a teenaged male who underwent liver biopsy for investigation of liver dysfunction of unknown cause. The diagnosis was not made until a re-biopsy approximately three years later, when the patient presented with recurrent skin lesions and elevated blood and urinary protoporphyrin levels. The liver biopsies contained brownish deposits that exhibited birefringence under polarized light and porphyrin fluorescence under fluorescence spectroscopy. EPP should be considered in young patients with unexplained liver dysfunction, skin symptoms, and seasonal changes in symptoms. Fluorescence spectroscopy of liver biopsy tissue can be a useful tool in the diagnosis of EPP.

Porcine embryo development and inactivation of microorganisms after ultraviolet-C irradiation at 228 nm.

It is important to prevent contamination inside the incubator as a method of preventing microbial infections during the embryo culture. In the present study, we examined the effects of ultraviolet-C (UV-C) irradiation, used for microorganism inactivation, on embryo development and the growth of bacteria, including Escherichia coli and Staphylococcus aureus, and the fungus Cladosporium cladosporioides. In the embryo irradiation experiment, we examined the effects of the plastic lid of the culture dish, irradiation distances (10, 20, and 25 cm), and different irradiation wavelengths (228 and 260 nm) during embryo culture for 7 days on the development and quality of porcine in vitro-fertilized embryos. None of the embryos cultured in dishes without plastic lids developed into blastocysts after irradiation with 228 nm UV-C. When porcine embryos were cultured in a culture dish with lids, the 228 nm UV-C irradiation decreased blastocyst formation rates of the embryos but not their quality, irrespective of the UV-C irradiation distance. Moreover, irradiation with 260 nm UV-C, even with plastic lids, had more detrimental effects on embryo development than irradiation with 228 nm UV-C. Investigation of the inactivating effects of UV-C irradiation at 228 nm and 260 nm on the growth of the bacteria and fungus showed that 260 nm UV-C reduced the viability to a greater extent than 228 nm UV-C. Moreover, the disinfection efficacy for the bacteria increased when the irradiation duration increased and the distance decreased. In conclusion, porcine embryos can develop into blastocysts without loss of quality even after continuous long-duration irradiation (7 days) with 228 nm UV-C, which can inactivate the growth of bacteria and the tested fungus; however, the development rate of the embryo is reduced.

High-sensitivity hyperspectral vibrational imaging of heart tissues by mid-infrared photothermal microscopy.

Visualizing the spatial distribution of chemical compositions in biological tissues is of great importance to study fundamental biological processes and origin of diseases. Raman microscopy, one of the label-free vibrational imaging techniques, has been employed for chemical characterization of tissues. However, the low sensitivity of Raman spectroscopy often requires a long acquisition time of Raman measurement or a high laser power, or both, which prevents one from investigating large-area tissues in a nondestructive manner. In this work, we demonstrated chemical imaging of heart tissues using mid-infrared photothermal (MIP) microscopy that simultaneously achieves the high sensitivity benefited from IR absorption of molecules and the high spatial resolution down to a few micrometers. We successfully visualized the distributions of different biomolecules, including proteins, phosphate-including proteins, and lipids/carbohydrates/amino acids. Further, we experimentally compared MIP microscopy with Raman microscopy to evaluate the sensitivity and photodamage to tissues. We proved that MIP microscopy is a highly sensitive technique for obtaining vibrational information of molecules in a broad fingerprint region, thereby it could be employed for biological and diagnostic applications, such as live-tissue imaging.

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.

Accumulation of Uroporphyrin I in Necrotic Tissues of Squamous Cell Carcinoma after Administration of 5-Aminolevulinic Acid.

5-aminolevulinic acid (5-ALA)-induced protoporphyrin IX (PpIX) fluorescence is widely used for the intraoperative detection of malignant tumors. However, the fluorescence emission profiles of the accompanying necrotic regions of these tumors have yet to be determined. To address this, we performed fluorescence and high-performance liquid chromatography (HPLC) analyses of necrotic tissues of squamous cancer after 5-ALA administration. In resected human lymph nodes of metastatic squamous cell carcinoma, we found a fluorescence peak at approximately 620 nm in necrotic lesions, which was distinct from the PpIX fluorescence peak at 635 nm for viable cancer lesions. Necrotic lesions obtained from a subcutaneous xenograft model of human B88 oral squamous cancer also emitted the characteristic fluorescence peak at 620 nm after light irradiation: the fluorescence intensity ratio (620 nm/635 nm) increased with the energy of the irradiation light. HPLC analysis revealed a high content ratio of uroporphyrin I (UPI)/total porphyrins in the necrotic cores of murine tumors, indicating that UPI is responsible for the 620 nm peak. UPI accumulation in necrotic tissues after 5-ALA administration was possibly due to the failure of the heme biosynthetic pathway. Taken together, fluorescence imaging of UPI after 5-ALA administration may be applicable for the evaluation of tumor necrosis.

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.

Hybrid optical imaging with near-infrared, mid-infrared, and terahertz wavelengths for nondestructive inspection [Invited].

Optical imaging is a powerful tool for nondestructive inspection, with high spatial resolution and low invasiveness. As light-material interactions vary a great deal depending on the wavelength, it is difficult to select the best imaging wavelength without prior knowledge of the optical properties of the material. To overcome this difficulty, we constructed a hybrid optical imaging system using three different wavelengths: near-infrared (NIR), mid-infrared (MIR), and terahertz (THz) regions. The same imaging optics were integrated with different light sources and detectors. Depending on the light-material interaction and detection sensitivity, NIR and THz imaging indicated some potential for nondestructive inspection, but MIR imaging showed difficulty. A combination of NIR and THz imaging will be a powerful tool for optical nondestructive inspection.

Quantitative evaluation of SARS-CoV-2 inactivation using a deep ultraviolet light-emitting diode.

Inactivation technology for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is certainly a critical measure to mitigate the spread of coronavirus disease 2019 (COVID-19). A deep ultraviolet light-emitting diode (DUV-LED) would be a promising candidate to inactivate SARS-CoV-2, based on the well-known antiviral effects of DUV on microorganisms and viruses. However, due to variations in the inactivation effects across different viruses, quantitative evaluations of the inactivation profile of SARS-CoV-2 by DUV-LED irradiation need to be performed. In the present study, we quantify the irradiation dose of DUV-LED necessary to inactivate SARS-CoV-2. For this purpose, we determined the culture media suitable for the irradiation of SARS-CoV-2 and optimized the irradiation apparatus using commercially available DUV-LEDs that operate at a center wavelength of 265, 280, or 300 nm. Under these conditions, we successfully analyzed the relationship between SARS-CoV-2 infectivity and the irradiation dose of the DUV-LEDs at each wavelength without irrelevant biological effects. In conclusion, total doses of 1.8 mJ/cm2 for 265 nm, 3.0 mJ/cm2 for 280 nm, and 23 mJ/cm2 for 300 nm are required to inactivate 99.9% of SARS-CoV-2. Our results provide quantitative antiviral effects of DUV irradiation on SARS-CoV-2, serving as basic knowledge of inactivation technologies against SARS-CoV-2.

Computationally image-corrected dual-comb microscopy with a free-running single-cavity dual-comb fiber laser.

Dual-comb microscopy (DCM), an interesting imaging modality based on the optical-frequency-comb (OFC) mode and image pixel one-to-one correspondence, benefits from scan-less full-field imaging and simultaneous confocal amplitude and phase imaging. However, the two fully frequency-stabilized OFC sources requirement hampers DCM practicality due to the complexity and costs. Here, a bidirectional single-cavity dual-comb fiber laser (SCDCFL) is adopted as a DCM low-complexity OFC source. Although the residual timing jitter in the SCDCFL blurs the image of a static object acquired by DCM, computational image correction significantly suppresses the image blur. Nanometer-order step surface profilometry with a 14.0 nm uncertainty highlights the computationally image-corrected DCM effectiveness. We further discuss a possibility to expand the computational image correction to a dynamic object and demonstrate its preliminary experiment. The proposed method enhances the DCM generality and practicality due to low-complexity OFC source.

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.