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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Refractive index sensing with temperature compensation by a multimode-interference fiber-based optical frequency comb sensing cavity.

We proposed a refractive index (RI) sensing method with temperature compensation by using an optical frequency comb (OFC) sensing cavity including a multimode-interference (MMI) fiber, namely, the MMI-OFC sensing cavity. The MMI-OFC sensing cavity enables simultaneous measurement of material-dependent RI and sample temperature by decoding from the comb spacing frequency shift and the wavelength shift of the OFC. We realized the simultaneous and continuous measurement of RI-related concentration of a liquid sample and its temperature with precisions of 1.6 × 10-4 RIU and 0.08 °C. The proposed method would be a useful means for the various applications based on RI sensing.

Visualization of internal structure and internal stress in visibly opaque objects using full-field phase-shifting terahertz digital holography.

We construct a full-field phase-shifting terahertz digital holography (PS-THz-DH) system by use of a THz quantum cascade laser and an uncooled, 2D micro-bolometer array. The PS-THz-DH enables us to separate the necessary diffraction-order image from unnecessary diffraction-order images without the need for spatial Fourier filtering, leading to suppress the decrease of spatial resolution. 3D shape of a visibly opaque object is visualized with a sub-millimeter lateral resolution and a sub-µm axial resolution. Also, the digital focusing of amplitude image enables the visualization of internal structure with the millimeter-order axial selectivity. Furthermore, the internal stress distribution of an externally compressed object is visualized from the phase image. The demonstrated results imply a possibility for non-destructive inspection of visibly opaque non-metal materials.

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.

Refractive-index-sensing optical comb based on photonic radio-frequency conversion with intracavity multi-mode interference fiber sensor.

Optical frequency combs (OFCs) have attracted attention as optical frequency rulers due to their tooth-like discrete spectra together with their inherent mode-locking nature and phase-locking control to a frequency standard. Based on this concept, their applications until now have been demonstrated in the fields of optical frequency metrology. However, if the utility of OFCs can be further expanded beyond their application by exploiting new aspects of OFCs, this will lead to new developments in optical metrology and instrumentation. Here, we report a fiber sensing application of OFCs based on a coherent link between the optical and radio frequencies, enabling high-precision refractive index measurement based on frequency measurement in radio-frequency (RF) region. Our technique encodes a refractive index change of a liquid sample into a repetition frequency of OFC by a combination of an intracavity multi-mode-interference fiber sensor and wavelength dispersion of a cavity fiber. Then, the change in refractive index is read out by measuring the repetition frequency in RF region based on a frequency standard. Use of an OFC as a photonic RF converter will lead to the development of new applications in high-precision fiber sensing with the help of functional fiber sensors and precise RF measurement.

Multicascade-linked synthetic wavelength digital holography using an optical-comb-referenced frequency synthesizer.

Digital holography (DH) is a promising method for non-contact surface topography because the reconstructed phase image can visualize the nanometer unevenness in a sample. However, the axial range of this method is limited to the range of the optical wavelength due to the phase wrapping ambiguity. Although the use of two different wavelengths of light and the resulting synthetic wavelength, i.e., synthetic wavelength DH, can expand the axial range up to several hundreds of millimeters, its axial precision does not reach sub-micrometer. In this article, we constructed a tunable external cavity laser diode phase-locked to an optical frequency comb, namely, an optical-comb-referenced frequency synthesizer, enabling us to generate multiple synthetic wavelengths within the range of 32 µm to 1.20 m. A multiple cascade link of the phase images among an optical wavelength ( = 1.520 µm) and 5 different synthetic wavelengths ( = 32.39 µm, 99.98 µm, 400.0 µm, 1003 µm, and 4021 µm) enables the shape measurement of a reflective millimeter-sized stepped surface with the axial resolution of 34 nm. The axial dynamic range, defined as the ratio of the axial range ( = 2.0 mm) to the axial resolution ( = 34 nm), achieves 5.9 × 105, which is larger than that of previous synthetic wavelength DH. Such a wide axial dynamic range capability will further expand the application field of DH for large objects with meter dimensions.

Scan-less hyperspectral dual-comb single-pixel-imaging in both amplitude and phase.

We have developed a hyperspectral imaging scheme that involves a combination of dual-comb spectroscopy and Hadamard-transform-based single-pixel imaging. The scheme enables us to obtain 12,000 hyperspectral images of amplitude and phase at a spatial resolution of 46 µm without mechanical scanning. The spectral resolution given by the data point interval in the frequency domain is 20 MHz and the comb mode interval is 100 MHz over a spectral range of 1.2 THz centered at 191.5 THz. As an initial demonstration of our scheme, we obtained spectroscopic images of a standard test chart through an etalon plate. The thickness of an absorptive chromium-coated layer on a float-glass substrate was determined to be 70 nm from the hyperspectral phase images in the near-infrared wavelength region.

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.

Label-free evaluation of myocardial infarction and its repair by spontaneous Raman spectroscopy.

Raman spectroscopy, which provides information about molecular species and structures of biomolecules via intrinsic molecular vibrations, can analyze physiological and pathological states of tissues on the basis of molecular constituents without staining. In this study, we analyzed Raman spectra of myocardial infarction and its repair in rats using the hypothesis that the myocardium in the course of myocardial infarction and its repair could be recognized by spontaneous Raman spectroscopy on the basis of chemical changes in myocardial tissues. Raman spectra were acquired from unfixed frozen cross sections of normal and infarcted heart tissues upon excitation at 532 nm. Raman spectra of the infarcted tissues were successfully obtained at characteristic time points: days 2, 5, and 21 after coronary ligation, at which the main components of the infarcted region were coagulation necrosis, granulation tissue, and fibrotic tissue, respectively. The latent variable weights calculated by a multivariate classification method, partial least-squares-discriminant analysis (PLS-DA), revealed fundamental information about the spectral differences among the types of tissues on the basis of molecular constituents. A prediction model for the evaluation of these tissue types was established via PLS-DA. Cross-validated sensitivities of 99.3, 95.3, 96.4, and 91.3% and specificities of 99.4, 99.5, 96.5, and 98.3% were attained for the discrimination of normal, necrotic, granulation, and fibrotic tissue, respectively. A two-dimensional image of a marginal area of infarction was successfully visualized via PLS-DA. Our results demonstrated that spontaneous Raman spectroscopy combined with PLS-DA is a novel label-free method of evaluating myocardial infarction and its repair.

Photoacoustic microscopy using ultrashort pulses with two different pulse durations.

We propose photoacoustic microscopy using ultrashort pulses with two different pulse durations in the range from femtoseconds to picoseconds. The subtraction of images for longer-pulse excitation from those for shorter-pulse excitation extracts two-photon photoacoustic images effectively, based on observation that the intensity ratio of two-photon to one-photon absorption-induced photoacoustic signals depends on the pulse duration in the same manner as the intensity ratio of two-photon and one-photon fluorescence signals. Two-photon photoacoustic microscopy using this subtraction method enables precise observation of the cross-sections of silicone hollows filled with the mixture of one-photon and two-photon absorption solutions.