Spiral interferometry acquires the topography of the sample and determines the elevation or depression of the sample structure by a single measurement. The method has advantages in simple measurements and stable optical setup due to the coaxial interferometer. However, the measurable axial range was limited by the wavelength of the light. Here, we demonstrate dual-wavelength spiral interferometry without a significant modification to the original setup of the spiral interferometry. The retrieved phase profile using a synthesized wavelength of 3.33 µm enlarges the measurable axial range with a magnification factor of 6.2 compared with that measured by the light at 532 nm.


Reduction of temperature drift in refractive-index-sensing optical frequency comb by active-dummy compensation of dual-comb configuration

IEEE/OSA Journal of Lightwave Technology


Wireless data transmission in the 560-GHz band utilizing terahertz wave generated through photomixing of a pair of distributed feedback lasers injection-locking to a Kerr micro-resonator soliton comb


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.


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


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


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.


Terahertz wireless communication in a 560-GHz band using a Kerr micro-resonator soliton comb


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.

International Journal of Molecular Sciences


Beam-angle-scanning surface plasmon resonance sensor for rapid, high-precision sensing of refractive index and bio-molecules


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 benzothiazolepyridinium 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-p-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.

Bioorganic & Medicinal Chemistry Letters


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


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


Quantitative evaluation of both histological and mechanical recovery in injured tendons using Fourier-transform second-harmonic-generation microscopy

IEEE Journal of Selected Topics in Quantum Electronics


Full-field fluorescence lifetime dual-comb microscopy using spectral mapping and frequency multiplexing of dual-comb optical beats

Fluorescence lifetime imaging microscopy (FLIM) is a powerful tool for quantitative fluorescence imaging because fluorescence lifetime is independent of concentration of fluorescent molecules or excitation/detection efficiency and is robust to photobleaching. However, since most FLIMs are based on point-to-point measurements, mechanical scanning of a focal spot is needed for forming an image, which hampers rapid imaging. Here, we demonstrate scan-less full-field FLIM based on a one-to-one correspondence between two-dimensional (2D) image pixels and frequency-multiplexed radio frequency (RF) signals. A vast number of dual-comb optical beats between dual optical frequency combs are effectively adopted for 2D spectral mapping and high-density frequency multiplexing in the RF region. Bimodal images of fluorescence amplitude and lifetime are obtained with high quantitativeness from amplitude and phase spectra of fluorescence RF comb modes without the need for mechanical scanning. The parallelized FLIM will be useful for rapid quantitative fluorescence imaging in life science.


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.


Synthesis and Optical Property of Quadrupolar Pyridinium Salt and Its Application as Bioimaging Agent


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


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.


Ultrasonic wave sensing using an optical-frequency-comb sensing cavity for photoacoustic imaging

We propose a novel ultrasonic wave sensing method employing an optical-frequency-comb (OFC) cavity, namely, an OFC sensing cavity-based ultrasonic wave sensor. We confirm the fundamental characteristics of the OFC sensing cavity-based ultrasonic wave sensor by using an ultrasonic wave transducer and realize the frequency response of up to 13 MHz with a linear response. We also confirm the potential applicability in photoacoustic imaging employing an 8-ns pulsed laser. Our demonstrations confirm the capability of the concept of the OFC sensing cavity in ultrasonic wave sensing and will also expand the potential of OFC technology.


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 provide an in situ method to non-invasively monitor osteoblastic collagen synthesis under mechanical stimulation. We applied second-harmonic-generation (SHG) microscopy to monitor the collagen fibers produced by osteoblast-like cells. To evaluate the influence of mechanical stimulation on collagen synthesis and maturation, we compared SHG images of osteoblast-produced collagen fibers with and without a cyclic stretch stimulus. Image analysis of the average SHG intensity indicated that the amount of osteoblastic collagen synthesis was significantly enhanced by the cyclic stretch. Furthermore, the maturity of the collagen fibers was not affected in the early stage of bone formation by the mechanical stimulus.© (2018) COPYRIGHT Society of Photo-Optical Instrumentation Engineers (SPIE). Downloading of the abstract is permitted for personal use only.


Scan-Less, Kilo-Pixel, Line-Field Confocal Phase Imaging with Spectrally Encoded Dual-Comb Microscopy

Confocal laser microscopy (CLM) is a powerful tool in life science research and industrial inspection, and its image acquisition rate is boosted by scan-less imaging techniques. However, the optical-intensity-based image contrast in CLM makes it difficult to visualize transparent non-fluorescent objects or reflective objects with nanometer unevenness. In this paper, we introduce an optical frequency comb (OFC) to scan-less CLM to give the optical-phase-based image contrast. One-dimensional (1D) image pixels of a sample are separately encoded onto OFC modes via 1D spectral encoding by using OFC as an optical carrier of amplitude and phase with a vast number of discrete frequency channels. Then, line-field confocal information of amplitude and phase are decoded from a mode-resolved OFC amplitude and phase spectra obtained by dual-comb spectroscopy without the need for mechanical scanning. The proposed confocal phase imaging will further expand the application fields of CLM.

IEEE Journal of Selected Topics in Quantum Electronics


Photonic-Crystal-Fiber-Coupled, Hand-Held, Polarization-Resolved Second-Harmonic-Generation Microscope for In Vivo Visualization of Dermal Collagen Fibers in Human Skin

Second-harmonic-generation (SHG) microscopy is a powerful tool for in vivo monitoring of collagen fibers in human skin. However, its practical use in the dermatological field is still limited due to the bulky and complicated setup. In this paper, we constructed a photonic-crystal-fiber-coupled, hand-held polarization-resolved SHG microscope for in vivo monitoring of collagen fibers in human skin. Fiber delivery of ultrashort pulse light was achieved by a large-mode-area photonic-crystal-fiber whereas the SHG microscopy setup was enclosed into a hand-held probe head. The combination of PCF with the hand-held probe head largely enhances the flexibility of measurement sites in the human skin.


Scan-less confocal phase imaging based on dual-comb microscopy

Confocal laser microscopy (CLM) is a powerful tool in life science research and industrial inspection because it offers two-dimensional optical sectioning or three-dimensional imaging capability with micrometer depth selectivity. Furthermore, scan-less imaging modality enables rapid image acquisition and high robustness against surrounding external disturbances in CLM. However, the objects to be measured must be reflective, absorptive, scattering, or fluorescent because the image contrast is given by the optical intensity. If a new image contrast can be provided by the optical phase, scan-less CLM can be further applied for transparent non-fluorescent objects or reflective objects with nanometer unevenness by providing information on refractive index, optical thickness, or geometrical shape. Here, we report scan-less confocal dual-comb microscopy offering a phase image in addition to an amplitude image with depth selectivity by using an optical frequency comb as an optical carrier of amplitude and phase with discrete ultra-multichannels. Our technique encodes confocal amplitude and phase images of a sample onto a series of discrete modes in the optical frequency comb with well-defined amplitude and phase to establish a one-to-one correspondence between image pixels and comb modes. The technique then decodes these images from comb modes with amplitude and phase. We demonstrate confocal phase imaging with milliradian phase resolution under micrometer depth selectivity on the millisecond timescale. As a proof of concept, we demonstrate the quantitative phase imaging of standing culture fixed cells and the surface topography of nanometer-scale step structures. Our technique for confocal phase imaging will find applications in three-dimensional visualization of stacked living cells in culture and nanometer surface topography of semiconductor objects.


Application of Scan-less Two-Dimensional Confocal Microscopy Based on a Combination of Confocal Slit With Wavelength/Space Conversion

Confocal laser microscope (CLM) has been widely used in the fields of the non-contact surface topography, biomedical imaging, and other applications, because the confocality gives two-dimensional (2D) optical-sectioning or three-dimensional (3D) imaging capability with the depth selectivity. Combination of line-focused CLM with one-dimensional (1D) spectral encoding CLM enables us to obtain the 2D confocal image without the need for the mechanical scanning. So-called scan-less 2D CLM is a unique imaging modality, however, there are no attempts to apply for practical application. In this paper, we constructed scan-less 2D CLM with the image acquisition time of 0.23 ms, the lateral resolution of 1.2 μm, the depth resolution of 2.4 μm, and apply it for different kinds of application to evaluate its practical potential.


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 µ to 1800 µ 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.


Texture analysis of secondharmonicgeneration images for quantitative analysis of reticular dermal collagen fibre in vivo in human facial cheek skin

Secondharmonicgeneration (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, noninvasiveness and highresolution threedimensional imaging. Although texture analysis of SHG images is a promising method for the quantitative analysis of wellorientated 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 (2DAC) analysis and twodimensional Fourier transform (2DFT), to evaluate the agedependent changes in reticular dermal collagen fibres in in vivo human cheek skin. Agedependent 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 2DAC analysis showed a significantly higher correlation with skin elasticity measured by a Cutometer®. These results clearly indicate that 2DAC analysis of SHG images is highly promising for the quantitative evaluation of agedependent 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.


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.


Quantitative Evaluation of Collagen Fiber Structure in Human Dermis Based on Two-Dimensional Auto-Correlation Analysis of SHG (Second Harmonic Generation) Image

SHG(第二高調波発生光)イメージの2次元自己相関解析に基づいたヒト真皮コラーゲン線維構造の定量化

Collagen fibers in the dermis are closely related to skin aging because collagen is one of structural proteins and hence plays an important role to determine the morphology and mechanical properties of the skin. Although histological and/or biochemical studies have been used to reveal the age-related decrease and disorganization of dermal collagen fibers, such invasive methods cannot be used for simple assessment or serial monitoring of skin aging. Therefore, there is a considerable need for non-invasive assessment method of skin aging. Recently, second-harmonic-generation (SHG) microscopy has attracted attention for in situ visualization of human dermal collagen fibers. Due to its specific collagen selectivity without the need for staining, non-invasiveness, and high-resolution 3D imaging capability, collagen fiber structure can be visualized as high-contrast SHG image in situ. However, extraction of quantitative parameters reflecting the collagen fiber structure from SHG image presents difficulties. In this article, we applied two-dimensional auto-correlation analysis to SHG images of dermal collagen fibers in human cheek skin. The collagen fiber structure index obtained from the analysis decreased significantly with aging. Furthermore, we confirmed a moderate correlation between the collagen fiber structure index and skin elasticity measured by a Cutometer®.This correlation indicates that the dermal collagen fiber structure plays an important role to determine skin elasticity. In other words, the auto-correlation analysis of SHG image can be used for evaluation of skin elasticity from the viewpoint of dermal collagen. The proposed method will be a powerful tool in the fields of skin cosmetics and anti-aging dermatology.

Transactions of the Japanese Society for Medical and Biological Engineering


Hand-held Second-harmonic-generation (SHG) Microscope for Dermatological Application

第2高調波発生顕微鏡の小型化

Second harmonic generation (SHG) microscopy allows in situ visualization of dermal collagen fibers in human skin due to its selectivity for collagen fiber. It is an attractive tool because it does not require staining, is non-invasiveness, and has high-resolution 3D imaging capability. However, its practical use in the dermatological field is still limited due to the bulky and complicated setup. Incorporation of fiber transmission of the laser light and a compact probe head into the microscope would enhance its compactness, robustness, flexibility, and hence convenience. In this study, we constructed a photonic-crystal-fiber (PCF) -coupled, hand-held SHG microscope for in situ monitoring of collagen fibers in human skin. By transmitting pulse light from a mode-locked Cr:Forsterite laser source via a large mode area PCF, it was possible to deliver pulse light with median wavelength of 1250nm and pulse width of 80 fs to the SHG microscope, without the need for external dispersion compensation. Components of a SHG microscope;galvano mirrors, relay lenses, objective lens, dichroic mirror, optical filters, and photon-counting photomultiplier, were mounted into a hand-held probe head (width=310mm, height=150mm, and depth=50mm), which is only 2% of the size of a conventional SHG microscope. We compared the imaging performance between the conventional SHG microscope and the hand-held SHG microscope, and confirmed comparable performance of the two. Finally, we demonstrated in situ visualization of collagen fibers in human skin using the hand-held SHG microscope with a compact probe head coupled to PCF. The hand-held SHG microscope greatly enhances the operability of in situ measurement of the human skin. The novel system will be a powerful tool for various dermatological applications.


Observation of Tendon Healing with Second-harmonic-generation (SHG) Microscopy

第2高調波発生光(SHG)顕微鏡を用いた腱修復の観察

Tendon rupture is a traumatic injury that is difficult to recover to the condition before injury. In previous studies, histological staining and tensile testing have been widely used to evaluate histological and mechanical healing. However, since both methods are destructive and invasive, it is difficult to apply these methods to clinical diagnosis. If the degree of healing can be visualized nondestructively and noninvasively, new findings may be obtained regarding mechanisms of the tendon healing process. In this study, we used second-harmonic-generation (SHG) microscopy to evaluate the degree of healing of ruptured tendon in a rabbit model. SHG microscopy has high selectivity and high image contrast with respect to the structural maturity, density, and aggregates of collagen molecule, without the need for histological sectioning and staining. Furthermore, since SHG light intensity sensitively reflects the structural maturity of collagen molecule and its aggregates, it has the potential to be a good indicator for the degree of healing of the injured tendon. By comparing the SHG images between 4-week-healing tendons and normal tendons in the animal model, we confirmed that SHG light intensity of the healing tendon was significantly lower than that of the normal tendon, indicating that the collagen structure in the healing tendon is still immature. Furthermore, we performed image analysis based on 2D Fourier transform of the acquired SHG images, and confirmed a significant difference in collagen distribution depending on the sample. These results indicate that SHG microscopy has the unique potential as an indicator of tendon healing.


This study aimed to evaluate the histological and mechanical features of tendon healing in a rabbit model with second-harmonic-generation (SHG) imaging and tensile testing. A total of eight male Japanese white rabbits were used for this study. The flexor digitorum tendons in their right leg were sharply transected, and then were repaired by intratendinous stitching. At four weeks post-operatively, the rabbits were killed and the flexor digitorum tendons in both right and left legs were excised and used as specimens for tendon healing (n = 8) and control (n = 8), respectively. Each specimen was examined by SHG imaging, followed by tensile testing, and the results of the two testing modalities were assessed for correlation. While the SHG light intensity of the healing tendon samples was significantly lower than that of the uninjured tendon samples, 2D Fourier transform SHG images showed a clear difference in collagen fibre structure between the uninjured and the healing samples, and among the healing samples. The mean intensity of the SHG image showed a moderate correlation (R(2) = 0.37) with Young's modulus obtained from the tensile testing. Our results indicate that SHG microscopy may be a potential indicator of tendon healing.Cite this article: E. Hase, K. Sato, D. Yonekura, T. Minamikawa, M. Takahashi, T. Yasui. Evaluation of the histological and mechanical features of tendon healing in a rabbit model with the use of second-harmonic-generation imaging and tensile testing. Bone Joint Res 2016;5:577-585. DOI: 10.1302/2046-3758.511.BJR-2016-0162.R1.


Scan-Less Full-Field Confocal Microscopy by a Combination of Confocal Slit with Wavelength/Space Conversion

スリット共焦点と波長/空間変換を用いたスキャンレス・フルフィールド共焦点顕微鏡

Journal of the Japan Society for Precision Engineering


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.


Optical-frequency-comb based ultrasound sensor

Progress in Biomedical Optics and Imaging - Proceedings of SPIE


Scan-less confocal phase imaging with dual comb microscopy


Orientation analysis of collagen fibers in healing tendon by using second-harmonic-generation microscopy


In situ monitoring of collagen fibers in human skin using a photonic-crystal-fiber-coupled, hand-held, second-harmonic-generation microscope