Shuichiro Fukushima

Ex vivo and in vivo second-harmonic-generation imaging of dermal collagen fiber in skin: comparison of imaging characteristics between mode-locked Cr:forsterite and Ti:sapphire lasers

Second-harmonic-generation (SHG) microscopy is an interesting new tool for observing dermal collagen fiber in skin. However, conventional SHG microscopy using a mode-locked Ti:sapphire laser suffers from low penetration depth and a slow image acquisition rate caused by scattering and absorption in tissue, making it difficult to use for in vivo applications on human skin. We develop an SHG microscope equipped with a mode-locked Cr:forsterite laser with a long wavelength and compare its imaging characteristics with that of a Ti:sapphire-laser-based SHG microscope for the measurement of dermal collagen fiber in animal and human skins. The results indicate the suitability of the Cr:forsterite laser-based SHG microscope for in vivo imaging of human skin.

Observation of dermal collagen fiber in wrinkled skin using polarization-resolved second-harmonic-generation microscopy

Optical probe methods for in vivo assessments of cutaneous photoaging are necessary in fields such as anti-aging dermatology and skin cosmetic development. We investigated the relation between wrinkle direction and collagen orientation in ultraviolet-B-exposed (UVB-exposed) skin using polarization-resolved second-harmonic-generation (SHG) microscopy. A polarization anisotropic image of the SHG light indicated that wrinkle direction in UVB-exposed skin is predominantly parallel to the orientation of dermal collagen fibers. Furthermore, collagen orientation in post-UVB-exposed skin with few wrinkles changed from that of UVB-exposed wrinkled skin to that of no-UVB-exposed skin. The method proposed has the potential to become a powerful non-invasive tool for assessment of cutaneous photoaging.

In vivo observation of age-related structural changes of dermal collagen in human facial skin using collagen-sensitive second harmonic generation microscope equipped with 1250-nm mode-locked Cr:Forsterite laser

Abstract. In vivo visualization of human skin aging is demonstrated using a Cr:Forsterite (Cr:F) laser-based, collagen-sensitive second harmonic generation (SHG) microscope. The deep penetration into human skin, as well as the specific sensitivity to collagen molecules, achieved by this microscope enables us to clearly visualize age-related structural changes of collagen fiber in the reticular dermis. Here we investigated intrinsic aging and/or photoaging in the male facial skin. Young subjects show dense distributions of thin collagen fibers, whereas elderly subjects show coarse distributions of thick collagen fibers. Furthermore, a comparison of SHG images between young and elderly subjects with and without a recent life history of excessive sun exposure show that a combination of photoaging with intrinsic aging significantly accelerates skin aging. We also perform image analysis based on two-dimensional Fourier transformation of the SHG images and extracted an aging parameter for human skin. The in vivo collagen-sensitive SHG microscope will be a powerful tool in fields such as cosmeceutical sciences and anti-aging dermatology.

In vivo visualization of dermal collagen fiber in skin burn by collagen-sensitive second-harmonic-generation microscopy

Abstract. Optical assessment of skin burns is possible with second-harmonic-generation (SHG) microscopy due to its high sensitivity to thermal denaturation of collagen molecules. In contrast to previous studies that were performed using excised tissue specimens ex vivo, in vivo observation of dermal collagen fibers in living rat burn models with SHG microscopy is demonstrated. Changes in signal vanishing patterns in the SHG images are confirmed to be dependent on the burn degree. Comparison of the SHG images with Masson’s trichrome-stained images indicated that the observed patterns were caused by the coexistence of molten and fibrous structures of dermal collagen fibers. Furthermore, a quantitative parameter for burn assessment based on the depth profile of the mean SHG intensity across the entire SHG image is proposed. These results and discussions imply a potential of SHG microscopy as a minimally invasive, highly quantitative tool for skin burn assessment.

Accumulation of advanced glycation end-products in human dentine.

Cross-linking of collagen by Advanced Glycation End-products (AGEs) occurs by non-enzymatic glycation (Maillard reaction). The purpose of this study was to examine whether AGEs are formed in human dentinal collagen, and to consider any possible influence of AGEs on dentinal physiology. Mechanical characteristics, fluorescence spectra and immunohistochemical analyses of demineralized dentine sections from young subjects were compared with those of aged ones. The same investigations were performed with young dentine artificially glycated by incubation in 0.1M ribose solution. Indentation measurement indicated that the sections from aged dentine were mechanically harder than those from young dentine. The hardness of young dentine increased after incubation in ribose solution. Fluorescence peak wavelength of the young dentine was shorter than that of the aged one, but shifted towards the peak wavelength of the aged one after incubation in ribose solution. These changes were considered to be due to accumulation of AGEs. Existence of AGEs in dentinal collagen was confirmed by immunohistochemical analysis. The obtained results suggest that AGEs accumulation occurs in dentinal collagen and is affected by both human age and physiological conditions such as glucose level in blood because dentinal collagen receives nourishment via dental pulp and tubules.

Motion-artifact-robust, polarization-resolved second-harmonic-generation microscopy based on rapid polarization switching with electro-optic Pockells cell and its application to in vivo visualization of collagen fiber orientation in human facial skin

Polarization-resolved second-harmonic-generation (PR-SHG) microscopy is a powerful tool for investigating collagen fiber orientation quantitatively with low invasiveness. However, the waiting time for the mechanical polarization rotation makes it too sensitive to motion artifacts and hence has hampered its use in various applications in vivo. In the work described in this article, we constructed a motion-artifact-robust, PR-SHG microscope based on rapid polarization switching at every pixel with an electro-optic Pockells cell (PC) in synchronization with step-wise raster scanning of the focus spot and alternate data acquisition of a vertical-polarization-resolved SHG signal and a horizontal-polarization-resolved one. The constructed PC-based PR-SHG microscope enabled us to visualize orientation mapping of dermal collagen fiber in human facial skin in vivo without the influence of motion artifacts. Furthermore, it implied the location and/or age dependence of the collagen fiber orientation in human facial skin. The robustness to motion artifacts in the collagen orientation measurement will expand the application scope of SHG microscopy in dermatology and collagen-related fields.

In vivo time-lapse imaging of skin burn wound healing using second-harmonic generation microscopy

Wound healing is a process to repair the damaged tissue caused by thermal burn, incised wound, or stab wound. Although the wound healing has many aspects, it is common for dynamics of collagen fiber, such as decomposition, production, or growth, to be closely related with wound healing. If such the healing process can be visualized as a timelapse image of the collagen fiber in the same subject, one may obtain new findings regarding biological repairing mechanisms in the healing process. In this article, to investigate the temporal modoification of dermal collagen fiber in the burn wound healing, we used second-harmonic-generation (SHG) microscopy, showing high selectivity and good image contrast to collagen molecules as well as high spatial resolution, optical three-dimensional sectioning, minimal invasiveness, deep penetration, the absence of interference from background light, and in vivo measurement without additional staining. Since SHG light arises from a non-centrosymmetric triple helix of three polypeptide chains in the collagen molecule, SHG intensity sensitively reflects the structure maturity of collagen molecule and its aggregates. A series of time-lapse SHG images during the wound healing process of 2 weeks clearly indicated that condensation and melting of dermal collagen fibers by the deep dermal burn, decomposition of the damaged collagen fibers in the inflammation phase, production of new collagen fibers in the proliferation phase, and the growth of the new collagen fibers in the remodeling phase. These results show a high potential of SHG microscopy for optical assessment of the wound healing process in vivo.

Decrease in fluorescence lifetime by glycation of collagen and its application in determining advanced glycation end-products in human dentin

Advanced Glycation End-products (AGEs) are produced by the Maillard reaction, which causes cross-linking of collagen and results in changes in the mechanical properties of collagen tissues. Several types of AGE fluoresce, and measurement of this fluorescence is effective for determining the presence of AGEs. Because fluorescence intensity by steady-state fluorometry is affected by sample surface condition and light source, we focused on fluorescence lifetime measurement (FLM). We found that fluorescence lifetime of collagen gel decreased with glycation progress. In vivo application of FLM for determination of AGEs was confirmed in human dentin.

Photo-induced cell damage analysis for multi-focus CARS microscopy

We investigated photo-induced cell damage for multi-focus CARS (coherent anti-Stokes Raman scattering) microscopy. In general, using a near-infrared pulse light source, photo-induced damage is dominantly caused via multi-photon induced phenomena, and the peak power of the excitation light is limited for the non-invasive imaging. We obtained cell viability images during single- or multi-focus (7 foci) exposure of which wavelength and pulse duration were 709 nm and 5 ps. The laser power of one focal spot was respectively set to 27.8 mW and 14.5 mW for single- and multi-focus excitation because those excitation beams induce the comparable signals for third-order nonlinear phenomena. The cell viability was observed using DAPI fluorophore that mainly stains DNA of dead cells. As a result, we found that the single-focus excitation with 27.8 mW/spot caused cell damage within 6 min. In contrast, photo-induced damage was not detected until 20 min for the multi-focus excitation with 14.5 mW/spot and 7 foci. The results suggest that the photo-induced damage is a serious problem on the single-focus excitation, and the multi-focus excitation method is preferable for CARS imaging.

Invited Article: Label-free nerve imaging with a coherent anti-Stokes Raman scattering rigid endoscope using two optical fibers for laser delivery

A coherent anti-Stokes Raman scattering (CARS) rigid endoscope using two optical fibers to deliver excitation beams individually is developed. The use of two optical fibers allows the correction of longitudinal chromatic aberration and enhances the CARS signal by a factor of 2.59. The endoscope is used to image rat sciatic nerves with an imaging time of 10 s. Imaging of the rabbit prostatic fascia without sample slicing is also demonstrated, which reveals the potential for the application of the CARS endoscope to robot-assisted surgery.A coherent anti-Stokes Raman scattering (CARS) rigid endoscope using two optical fibers to deliver excitation beams individually is developed. The use of two optical fibers allows the correction of longitudinal chromatic aberration and enhances the CARS signal by a factor of 2.59. The endoscope is used to image rat sciatic nerves with an imaging time of 10 s. Imaging of the rabbit prostatic fascia without sample slicing is also demonstrated, which reveals the potential for the application of the CARS endoscope to robot-assisted surgery.