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. Furthermore, polarization-resolved SHG microscopy can provide additional insights regarding the direction of collagen fibers, i.e., collagen fiber orientation. However, their 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 without significant change of the linear polarization 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

Nanometer-precision surface metrology of millimeter-size 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 surface profilometry of objects with sub-wavelength precision. In this article, we demonstrate full-cascade-linked synthetic-wavelength DH (FCL-SW-DH) for nanometer-precision surface metrology of millimeter-size 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 (EOM-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 um to 29.7 mm. We determine the 0.1000mm-stepped surface with axial uncertainty of 6.1 nm within the maximum axial range of 14.85 mm.Comment: 22 pages, 6 figure

Evaluation of the histological and mechanical features of tendon healing in a rabbit model

Objectives 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. Materials and Methods 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. Results 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 (R2 = 0.37) with Young’s modulus obtained from the tensile testing. Conclusion Our results indicate that SHG microscopy may be a potential indicator of tendon healing

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

Scan-less full-field fluorescence-lifetime dual-comb microscopy using two-dimensional spectral mapping and frequency multiplexing of dual-optical-comb 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 FLIM is based on point-to-point measurements, mechanical scanning of a focal spot is needed for forming an image, which hampers rapid imaging. In this article, we demonstrate scan-less full-field FLIM based on a one-to-one correspondence between two-dimensional (2D) image pixels and frequency-multiplexed RF signals. A vast number of dual-optical-comb beats between dual optical frequency combs is effectively adopted for 2D spectral mapping and high-density frequency multiplexing in radio-frequency 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 proposed method will be useful for rapid quantitative fluorescence imaging in life science.Comment: 38 pages, 8 figures, 1 tabl

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

Optical image amplification in dualcomb 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

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

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. The proposed confocal phase imaging will further expand the application fields of CLM