Raman Spectroscopic Evaluation of Composition of Matrix Synthesized by Osteoblasts under Microvibration Stimulation


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.


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


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.


Major target for UV-induced complete loss of HIV-1 infectivity: A model study of single-stranded RNA enveloped viruses


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.


Establishment of an epicutaneously sensitized murine model of shellfish allergy and evaluation of skin condition by Raman microscopy.


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


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 lightmaterial interactions vary a great deal depending on the wavelength, it is difficult to select the best imag- ing 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 lightmaterial 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.

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.


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


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.


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.


Adaptive-sampling near-Doppler-limited terahertz dual-comb spectroscopy with a free-running single-cavity fiber laser

Dual-comb spectroscopy (DCS) is an emerging spectroscopic tool with the potential to simultaneously achieve a broad spectral coverage and ultrahigh spectral resolution with rapid data acquisition. However, the need for two independently stabilized ultrafast lasers significantly hampers the potential application of DCS. We demonstrate mode-resolved DCS in the THz region based on a free-running single-cavity dual-comb fiber laser with the adaptive sampling method. While the use of a free-running single-cavity dual-comb fiber laser eliminates the need for two mode-locked lasers and their frequency control, the adaptive sampling method strongly prevents the degradation of spectroscopic performance caused by the residual timing jitter in the free-running dual-comb laser. Doppler-limit-approaching absorption features with linewidths down to 25 MHz are investigated for low-pressure acetonitrile/air mixed gas by comb-mode-resolved THz spectroscopy. The successful demonstration clearly indicates its great potential for the realization of low-complexity, Doppler-limited THz spectroscopy instrumentation.


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.


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.


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 × 104 RIU and 0.08 °C. The proposed method would be a useful means for the various applications based on RI sensing.


Dual-comb spectroscopy (DCS) is useful for gas spectroscopy due to the high potential of optical frequency comb (OFC). However, fast Fourier transform (FFT) calculation of a huge amount of temporal data spends significantly longer time than the acquisition time of an interferogram. In this article, we demonstrate frequency-domain DCS by a combination of DCS with lock-in detection, namely LID-DCS. LID-DCS directly extracts an arbitrary OFC mode from a vast number of OFC modes without the need for FFT calculation. Usefulness of LID-DCS is demonstrated in the rapid monitoring of transient signal change and spectroscopy of hydrogen cyanide gas.


Improvement of dynamic range and repeatability in a refractive-index- sensing optical comb by combining saturable-absorber-mirror mode- locking with an intracavity multimode interference fiber sensor

A mode-locked fiber comb equipped with a multimode interference fiber sensor functions as a high-precision refractive-index (RI) sensor benefitting from precise RF measurement. However, its dynamic range and repeatability are hampered by the inherent characteristics of nonlinear- polarization-rotation mode-locking oscillation. In this article, we introduce saturable-absorber-mirror mode-locking for RI sensing with a wide dynamic range and high repeatability. While the RI dynamic range was expanded to 41.4 dB due to high robustness against cavity disturbance, the self-starting capability without the need for polarization control improves the RI sensing repeatability to 1.10 × 108 for each mode-locking activation. The improved dynamic range and repeatability will be useful for enhancing the performance of RI sensing.


Lens-less fiber coupling of a 1550-nm mode-locked fiber laser light on a low-temperature-grown GaAs photoconductive antenna


Preparation of hierarchically assembled silver nanostructures based on the morphologies of crystalline peptide-silver(I) complexes


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


Label-free Evaluation of Myocardial Infarct in Surgically Excised Ventricular Myocardium by Raman Spectroscopy.

Understanding the viability of the ischemic myocardial tissue is a critical issue in determining the appropriate surgical procedure for patients with chronic heart failure after myocardial infarction (MI). Conventional MI evaluation methods are; however, preoperatively performed and/or give an indirect information of myocardial viability such as shape, color, and blood flow. In this study, we realize the evaluation of MI in patients undergoing cardiac surgery by Raman spectroscopy under label-free conditions, which is based on intrinsic molecular constituents related to myocardial viability. We identify key signatures of Raman spectra for the evaluation of myocardial viability by evaluating the infarct border zone myocardium that were excised from five patients under surgical ventricular restoration. We also obtain a prediction model to differentiate the infarcted myocardium from the non-infarcted myocardium by applying partial least squares regression-discriminant analysis (PLS-DA) to the Raman spectra. Our prediction model enables identification of the infarcted tissues and the non-infarcted tissues with sensitivities of 99.98% and 99.92%, respectively. Furthermore, the prediction model of the Raman images of the infarct border zone enabled us to visualize boundaries between these distinct regions. Our novel application of Raman spectroscopy to the human heart would be a useful means for the detection of myocardial viability during surgery.


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 × 10, 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.


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.


Dual terahertz comb spectroscopy with a single free-running fibre laser

Dual terahertz (THz) comb spectroscopy enables high spectral resolution, high spectral accuracy, and broad spectral coverage; however, the requirement for dual stabilized femtosecond lasers hampers its versatility. We here report the first demonstration of dual THz comb spectroscopy using a single free-running fibre laser. By tuning the cavity-loss-dependent gain profile with an intracavity Lyot filter together with precise management of the cavity length and dispersion, dual-wavelength comb light beams with slightly detuned repetition frequencies are generated in a single laser cavity. Due to sharing of the same cavity, such comb light beams suffer from common-mode fluctuation of the repetition frequency, and hence the corresponding frequency difference between them is passively stable around a few hundred hertz within millihertz fluctuation. While greatly reducing the size, complexity, and cost of the laser source by use of a single free-running fibre laser, the dual THz comb spectroscopy system maintains a spectral bandwidth and dynamic range of spectral power comparable to a system equipped with dual stabilized fibre lasers, and can be effectively applied to high-precision spectroscopy of acetonitrile gas at atmospheric pressure. The demonstrated results indicate that this system is an attractive solution for practical applications of THz spectroscopy and other applications.


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.


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.


Real-Time Amplitude and Phase Imaging of Optically Opaque Objects by Combining Full-Field Off-Axis Terahertz Digital Holography with Angular Spectrum Reconstruction

Terahertz digital holography (THz-DH) has the potential to be used for non-destructive inspection of visibly opaque soft materials due to its good immunity to optical scattering and absorption. Although previous research on full-field off-axis THz-DH has usually been performed using Fresnel diffraction reconstruction, its minimum reconstruction distance occasionally prevents a sample from being placed near a THz imager to increase the signal-to-noise ratio in the hologram. In this article, we apply the angular spectrum method (ASM) for wavefront reconstruction in full-filed off-axis THz-DH because ASM is more accurate at short reconstruction distances. We demonstrate real-time phase imaging of a visibly opaque plastic sample with a phase resolution power of λ/49 at a frame rate of 3.5 Hz in addition to real-time amplitude imaging. We also perform digital focusing of the amplitude image for the same object with a depth selectivity of 447 μm. Furthermore, 3D imaging of visibly opaque silicon objects was achieved with a depth precision of 1.7 μm. The demonstrated results indicate the high potential of the proposed method for in-line or in-process non-destructive inspection of soft materials.

Journal of Infrared, Millimeter and Terahertz Waves


Photo-induced cell damage analysis for single- and multi-focus coherent anti-Stokes Raman scattering microscopy


Analytical imaging of colour pigments used in Japanese woodblock prints using Raman microspectroscopy


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.


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.


Efficient fluorescence detection of protoporphyrin IX in metastatic lymph nodes of murine colorectal cancer stained with indigo carmine.

Protoporphyrin IX (PpIX), a biochemical converted from 5-aminolevulinc acid (5-ALA) in living cells, is useful for intraoperative fluorescent detection of cancer metastasis in lymph nodes (LNs). However, unknown is whether the fluorescence of PpIX can be detected in the LNs when they coexist with indigo carmine, a blue dye commonly used for identification of sentinel LNs during surgery. To address this issue, we sought to evaluate the diagnostic usefulness of PpIX fluorescence in the presence of indigo carmine in a mouse LN metastasis model of rectal cancer after administration of 5-ALA. Spectral analysis of pure chemicals revealed that the absorption spectrum of indigo carmine widely overlapped with the fluorescence spectrum of PpIX specifically at the peak of 632nm, a common emission wavelength for detecting PpIX, but not at the other peak of 700nm. Due to such spectral overlap, the PpIX fluorescence intensity was significantly attenuated by mixture with indigo carmine at 632nm, but not at 700nm. Accordingly, fluorescent measurements of the mouse metastatic LN revealed more intense presentation of PpIX at 700nm than at 632nm, indicating that the diagnostic usefulness is greater at 700nm than at 632nm for the indigo carmine-dyed LNs after administration of 5-ALA. From these observations, we propose that the fluorescence measurement is more efficient at 700nm than at 632nm for detection of PpIX in metastatic LNs stained with indigo carmine.


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.

Transactions of the Japanese Society for Medical and Biological Engineering


Measurement of absolute frequency of continuous-wave terahertz radiation in real time using a free-running, dual-wavelength mode-locked, erbium-doped fibre laser.

A single, free-running, dual-wavelength mode-locked, erbium-doped fibre laser was exploited to measure the absolute frequency of continuous-wave terahertz (CW-THz) radiation in real time using dual THz combs of photo-carriers (dual PC-THz combs). Two independent mode-locked laser beams with different wavelengths and different repetition frequencies were generated from this laser and were used to generate dual PC-THz combs having different frequency spacings in photoconductive antennae. Based on the dual PC-THz combs, the absolute frequency of CW-THz radiation was determined with a relative precision of 1.2 × 10(-9) and a relative accuracy of 1.4 × 10(-9) at a sampling rate of 100 Hz. Real-time determination of the absolute frequency of CW-THz radiation varying over a few tens of GHz was also demonstrated. Use of a single dual-wavelength mode-locked fibre laser, in place of dual mode-locked lasers, greatly reduced the size, complexity, and cost of the measurement system while maintaining the real-time capability and high measurement precision.


Label-free detection of myocardial ischaemia in the perfused rat heart by spontaneous Raman spectroscopy.

Raman spectroscopy, which identifies intrinsic molecular constituents, has a potential for determining myocardial viability under label-free conditions. However, its suitability for evaluating myocardial ischaemia is undetermined. Focusing on cytochromes, i.e., representative molecules reflecting mitochondrial activity, we tested whether Raman spectroscopy is applicable for evaluating myocardial ischaemia especially during early ischaemic phase. We obtained spontaneous Raman spectra of the subepicardial myocardium in the Langendorff-perfused rat heart upon 532-nm excitation before and during the "stopped-flow," global ischaemia. Semi-quantitative values of the peak intensities at 750 and 1127 cm(-1), which reflect reduced cytochromes c and b, increased immediately and progressively after induction of the stopped flow, indicating progressive reduction of the mitochondrial respiration. Such spectral changes emerged before the loss of 1) mitochondrial membrane potentials measured by the fluorescence intensity of tetramethyl rhodamine ethyl ester or 2) staining of the triphenyl tetrazolium chloride dye in the myocardium. The progressive increases in the Raman peaks by stopped flow were significantly retarded by ischaemic preconditioning. Sequential measurements of the peak intensities at 750 and 1127 cm(-1) enabled early detection of the myocardial ischaemia based on the mitochondrial functions. These data suggest that Raman spectroscopy offers the potential to evaluate acute ischaemic heart under label-free conditions.


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


Dynamic terahertz spectroscopy of gas molecules mixed with unwanted aerosol under atmospheric pressure using fibre-based asynchronous-optical-sampling terahertz time-domain spectroscopy.

Terahertz (THz) spectroscopy is a promising method for analysing polar gas molecules mixed with unwanted aerosols due to its ability to obtain spectral fingerprints of rotational transition and immunity to aerosol scattering. In this article, dynamic THz spectroscopy of acetonitrile (CH3CN) gas was performed in the presence of smoke under the atmospheric pressure using a fibre-based, asynchronous-optical-sampling THz time-domain spectrometer. To match THz spectral signatures of gas molecules at atmospheric pressure, the spectral resolution was optimized to 1 GHz with a measurement rate of 1 Hz. The spectral overlapping of closely packed absorption lines significantly boosted the detection limit to 200 ppm when considering all the spectral contributions of the numerous absorption lines from 0.2 THz to 1 THz. Temporal changes of the CH3CN gas concentration were monitored under the smoky condition at the atmospheric pressure during volatilization of CH3CN droplets and the following diffusion of the volatilized CH3CN gas without the influence of scattering or absorption by the smoke. This system will be a powerful tool for real-time monitoring of target gases in practical applications of gas analysis in the atmospheric pressure, such as combustion processes or fire accident.


Simplified and optimized multispectral imaging for 5-ALA-based fluorescence diagnosis of malignant lesions.

5-aminolevulinic acid (5-ALA)-based fluorescence diagnosis is now clinically applied for accurate and ultrarapid diagnosis of malignant lesions such as lymph node metastasis during surgery. 5-ALA-based diagnosis evaluates fluorescence intensity of a fluorescent metabolite of 5-ALA, protoporphyrin IX (PPIX); however, the fluorescence of PPIX is often affected by autofluorescence of tissue chromophores, such as collagen and flavins. In this study, we demonstrated PPIX fluorescence estimation with autofluorescence elimination for 5-ALA-based fluorescence diagnosis of malignant lesions by simplified and optimized multispectral imaging. We computationally optimized observation wavelength regions for the estimation of PPIX fluorescence in terms of minimizing prediction error of PPIX fluorescence intensity in the presence of typical chromophores, collagen and flavins. By using the fluorescence intensities of the optimized wavelength regions, we verified quantitative detection of PPIX fluorescence by using chemical mixtures of PPIX, flavins, and collagen. Furthermore, we demonstrated detection capability by using metastatic and non-metastatic lymph nodes of colorectal cancer patients. These results suggest the potential and usefulness of the background-free estimation method of PPIX fluorescence for 5-ALA-based fluorescence diagnosis of malignant lesions, and we expect this method to be beneficial for intraoperative and rapid cancer diagnosis.


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.


Terahertz frequency-domain spectroscopy of low-pressure acetonitrile gas by a Photomixing terahertz synthesizer referenced to dual optical frequency combs

Journal of Infrared, Millimeter and Terahertz Waves


Highly sensitive fluorescence detection of metastatic lymph nodes of gastric cancer with photo-oxidation of protoporphyrin IX.

We established a highly sensitive and specific 5-ALA-induced fluorescence imaging method applicable in clinical settings. The novel method has a potential to become a useful tool for intraoperative rapid diagnosis of LN metastasis.

Specific visualization of PpIX was achieved in in vitro study. The method allowed highly sensitive detection of metastatic LNs, with sensitivity of 91.9% and specificity of 90.8% in the in vivo clinical trial. Receiver operating characteristic analysis indicated high diagnostic accuracy, with the area under the curve of 0.926.

EJSO - European Journal of Surgical Oncology


Development of molecular distribution analysis method of color pigments on Japanese woodblock prints by Raman spectral-imagin

ラマン散乱分光イメージング法による多色摺木版画の色材分子分布解析法の開発

Ukiyo-e prints, Japanese multicolor woodblock prints, is a traditional Japanese art that has been flourished since Edo period. To surmise the production technologies of woodblock prints, such as materials, carving technologies, and printing technologies, detailed analysis of color pigments that are one of the most important essential materials used in woodblock prints is required. In this study, we proposed a noninvasive analytical method of color pigments by means of Raman spectral-imaging. Pigment species and its molecular structures were clarified via Raman spectra of pigments. Raman imaging of woodblock prints revealed pigment distribution in plane and cross-section, which provided adhesion states of pigments on paper fibers. We also visualized a different pigment distribution on paper depending on glue content in pigment mixture. Our results indicated that Raman spectral-imaging would be a promising technique for evaluating a production technologies of ancient ukiyo-e prints that was still unknown at present.

錦絵をはじめとする多色摺木版画において，色材は木版画を特徴付ける重要な要素である．そのため，色材の分子構造および分子分布の詳細な解析が実現されれば，江戸時代の錦絵制作過程の重要な一部を推察することができる可能性がある．そこで本研究では，分子構造を強く反映した情報が得られるラマン散乱分光法に着目し，色材の分子構造と空間分布を同時に解析が可能な非侵襲的色材解析法の開発を行った．その結果，ラマン散乱分光法を用いることで色材の分子構造解析およびその空間分布を可視化することに成功した．特に，紙面内イメージングから紙の繊維上の色材分布を可視化することに成功した．また，紙面と垂直方向のイメージングから，色材の立体的空間分布を可視化することに成功した．さらに，膠含有/不含色材の空間分布の違いも明らかにし，色材分布に基づく摺刷技術解析の可能性を示した．以上の結果から，本手法は多色摺木版画制作技法の新たな解析手法となると考えられる．

Journal of Japan Society of Information and Knowledge


Real-time determination of absolute frequency in continuous-wave terahertz radiation with a photocarrier terahertz frequency comb induced by an unstabilized femtosecond laser


Ex vivo peripheral nerve detection of rats by spontaneous Raman spectroscopy.

Nerve-sparing surgery is increasingly being applied to avoid functional deficits of the limbs and organs following surgery. Peripheral nerves that should be preserved are, however, sometimes misidentified due to similarity of shape and color to non-nerve tissues. To avoid misidentification of peripheral nerves, development of an in situ nerve detection method is desired. In this study, we report the label-free detection of ex vivo peripheral nerves of Wistar rats by using Raman spectroscopy. We obtained Raman spectra of peripheral nerves (myelinated and unmyelinated nerves) and their adjacent tissues of Wistar rats without any treatment such as fixation and/or staining. For the identification of tissue species and further analysis of spectral features, we proposed a principal component regression-based discriminant analysis with representative Raman spectra of peripheral nerves and their adjacent tissues. Our prediction model selectively detected myelinated nerves and unmyelinated nerves of Wistar rats with respective sensitivities of 95.5% and 88.3% and specificities of 99.4% and 93.5%. Furthermore, important spectral features for the identification of tissue species were revealed by detailed analysis of principal components of representative Raman spectra of tissues. Our proposed approach may provide a unique and powerful tool for peripheral nerve detection for nerve-sparing surgery in the future.


Spectral fingerprinting of individual cells visualized by cavity-reflection-enhanced light-absorption microscopy.

The absorption spectrum of light is known to be a "molecular fingerprint" that enables analysis of the molecular type and its amount. It would be useful to measure the absorption spectrum in single cell in order to investigate the cellular status. However, cells are too thin for their absorption spectrum to be measured. In this study, we developed an optical-cavity-enhanced absorption spectroscopic microscopy method for two-dimensional absorption imaging. The light absorption is enhanced by an optical cavity system, which allows the detection of the absorption spectrum with samples having an optical path length as small as 10 m, at a subcellular spatial resolution. Principal component analysis of various types of cultured mammalian cells indicates absorption-based cellular diversity. Interestingly, this diversity is observed among not only different species but also identical cell types. Furthermore, this microscopy technique allows us to observe frozen sections of tissue samples without any staining and is capable of label-free biopsy. Thus, our microscopy method opens the door for imaging the absorption spectra of biological samples and thereby detecting the individuality of cells.


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.


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.


Detection of lymph node metastases in human colorectal cancer by using 5-aminolevulinic acid-induced protoporphyrin IX fluorescence with spectral unmixing.

Accurate evaluation of metastatic lymph nodes (LNs) is indispensable for adequate treatment of colorectal cancer (CRC) patients. Here, we demonstrate detection of metastases of human CRC in removed fresh LNs using 5-aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) fluorescence. A spectral unmixing method was employed to reduce the overlap of collagen autofluorescence on PpIX fluorescence. A total of 17 surgery patients with advanced CRC were included in this study. After 5-ALA at a dose of 15 mg/kg of body weight was applied orally 2 h prior to surgery, 87 LNs were subjected to spectral fluorescence imaging and histopathological diagnosis, and statistical analysis was performed. No apparent side effect was observed to be associated with 5-ALA administration. The spectral unmixing fluorescence intensity of PpIX in metastatic LNs was 10.2-fold greater than that in nonmetastaic LNs. The receiver-operating-characteristic (ROC) analysis showed that the area under the curve (AUC) was calculated as 0.95. Our results show the potential of 5-ALA-induced PpIX fluorescence processed by spectral unmixing for detecting metastases in excised fresh LNs from patients with CRC, suggesting that this rapid and feasible method is applicable to gross evaluation of resected LN samples in pathology laboratories.

International Journal of Molecular Sciences


Fast spectral coherent anti-Stokes Raman scattering microscopy with high-speed tunable picosecond laser.

We develop a coherent anti-Stokes Raman scattering (CARS) microscopy system equipped with a tunable picosecond laser for high-speed wavelength scanning. An acousto-optic tunable filter (AOTF) is integrated in the laser cavity to enable wavelength scanning by varying the radio frequency waves applied to the AOTF crystal. An end mirror attached on a piezoelectric actuator and a pair of parallel plates driven by galvanometer motors are also introduced into the cavity to compensate for changes in the cavity length during wavelength scanning to allow synchronization with another picosecond laser. We demonstrate fast spectral imaging of 3T3-L1 adipocytes every 5 cm-1 in the Raman spectral region around 2850 cm-1 with an image acquisition time of 120 ms. We also demonstrate fast switching of Raman shifts between 2100 and 2850 cm-1, corresponding to CD2 symmetric stretching and CH2 symmetric stretching vibrations, respectively. The fast-switching CARS images reveal different locations of recrystallized deuterated and nondeuterated stearic acid.


Label-free detection of peripheral nerve tissues against adjacent tissues by spontaneous Raman microspectroscopy.

Detection of peripheral nerve tissues during surgery is required to avoid neural disturbance following surgery as an aspect of realizing better functional outcome. We provide a proof-of-principle demonstration of a label-free detection technique of peripheral nerve tissues, including myelinated and unmyelinated nerves, against adjacent tissues that employ spontaneous Raman microspectroscopy. To investigate the Raman spectral features of peripheral nerves in detail, we used unfixed sectioned samples. Raman spectra of myelinated nerve, unmyelinated nerve, fibrous connective tissue, skeletal muscle, tunica media of blood vessel, and adipose tissue of Wistar rats were analyzed, and Raman images of the tissue distribution were constructed using the map of the ordinary least squares regression (OLSR) estimates. We found that nerve tissues exhibited a specific Raman spectrum arising from axon or myelin sheath, and that the nerve tissues can be selectively detected against the other tissues. Moreover, myelinated and unmyelinated nerves can be distinguished by the intensity differences of 2,855 cm⁻¹, and 2,945 cm⁻¹, which are mainly derived from lipid and protein contents of nerve fibers. We applied this method to unfixed section samples of human periprostatic tissues excised from prostatic cancer patients. Myelinated nerves, unmyelinated nerves, fibrous connective tissues, and adipose tissues of the periprostatic tissues were separately detected by OLSR analysis. These results suggest the potential of the Raman spectroscopic observation for noninvasive and label-free nerve detection, and we expect this method could be a key technique for nerve-sparing surgery.


Real-time imaging of laser-induced membrane disruption of a living cell observed with multifocus coherent anti-Stokes Raman scattering microscopy.

We demonstrate the real-time imaging of laser-induced disruption of the cellular membrane in a living HeLa cell and its cellular response with a multifocus coherent anti-Stokes Raman scattering (CARS) microscope. A near-infrared pulsed laser beam tightly focused on the cellular membrane of a living cell induces ablation at the focal point causing a local disruption of the cellular membrane. After the membrane disruption a dark spot decreasing CARS intensity of 2840 cm(-1) Raman shift at the disrupted site appears. This dark spot immediately disappears and a strong CARS signal is observed around the disrupted site. This increase of the CARS signal might be caused by resealing of the disrupted site via aggregation of the patch lipid vesicles in the cytoplasm. The accumulation of lipids around the disrupted site is also confirmed with three-dimensional CARS images of a cell before and after membrane disruption. The temporal behavior of the CARS signal at the disrupted site is observed to detect the fusion dynamics of patch vesicles.


Multi-focus excitation coherent anti-Stokes Raman scattering (CARS) microscopy and its applications for real-time imaging.

We developed a multi-focus excitation coherent anti-Stokes Raman scattering (CARS) microscope using a microlens array scanner for real-time molecular imaging. Parallel exposure of a specimen with light from two highly controlled picosecond mode-locked lasers (jitter of 30 fs through an electronic low-pass filter with 150 Hz bandwidth, point-by-point wavelength scan within 300 ms) and parallel detection with an image sensor enabled real-time imaging. We demonstrated real-time CARS imaging of polystyrene beads (frame rate of 30 fps), a giant multi-lamellar vesicle of dipalmitoylphosphatidylcholine (frame rate of 10 fps), and living HeLa cells (frame rate of 10 fps).


Optical-frequency-comb based ultrasound sensor

Progress in Biomedical Optics and Imaging - Proceedings of SPIE


Hyperspectral single-pixel imaging with dual optical combs


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


Raman spectroscopic detection of peripheral nerves towards nerve-sparing surgery


Simple and optimum background-free estimation method of PPIX fluorescence for 5-ALA-based fluorescence diagnosis of malignant lesions


Molecular orientation imaging of liquid crystals by tunable-polarization-mode coherent anti-Stokes Raman scattering microscopy

We have developed a tunable-polarization-mode coherent anti-Stokes Raman scattering (CARS) microscope with compact polarization mode converters constructed using eight-segmented liquid-crystal spatial light modulators. The polarization modes, such as linear, radial, and azimuthal polarizations, of two excitation beams are controlled independently and are switched without any mechanical tuning in less than 300 ms. We use the system to detect the molecular orientation of 4-cyano-4'-octylbiphenyl (8CB) liquid crystals aligned parallel and perpendicular to the optical axis. We also observe CARS images of liquid crystal defects known as focal conic domains, demonstrating the potential of our molecular orientation imaging system. (C) 2013 The Japan Society of Applied Physics


Jitter reduction of two synchronized picosecond mode-locked lasers using balanced cross-correlator with two-photon detectors

The authors have developed a highly synchronized picosecond mode-locked laser system. A balanced cross-correlator using two-photon detectors was employed to observe femtosecond order timing jitter between two picosecond lasers (1.26 fs with 150 Hz bandwidth and 7.14 fs with 1 kHz bandwidth), and a signal from the correlator was used as a feedback control signal to reduce the timing jitter. The timing jitter between the two lasers was reduced to 8 fs through a low-pass filter with 150 Hz bandwidth.