Discontinuity third harmonic generation microscopy for label-free imaging and quantification of intraepidermal nerve fibers.

Label-free imaging methodologies for nerve fibers rely on spatial signal continuity to identify fibers and fail to image free intraepidermal nerve endings (FINEs). Here, we present an imaging methodology-called discontinuity third harmonic generation (THG) microscopy (dTHGM)-that detects three-dimensional discontinuities in THG signals as the contrast. We describe the mechanism and design of dTHGM and apply it to reveal the bead-string characteristics of unmyelinated FINEs. We confirmed the label-free capability of dTHGM through a comparison study with the PGP9.5 immunohistochemical staining slides and a longitudinal spared nerve injury study. An intraepidermal nerve fiber (IENF) index based on a discontinuous-dot-connecting algorithm was developed to facilitate clinical applications of dTHGM. A preliminary clinical study confirmed that the IENF index was highly correlated with skin-biopsy-based IENF density (Pearson's correlation coefficient R = 0.98) and could achieve differential identification of small-fiber neuropathy (p = 0.0102) in patients with diabetic peripheral neuropathy.

Characterization of picosecond laser-induced optical breakdown using harmonic generation microscopy.

BACKGROUND AND OBJECTIVES: By creating microinjuries usually confined to the epidermis, a fractional picosecond 1064-nm Nd:YAG laser that delivers an array of highly focused beamlets can be effectively used for facial rejuvenation or resurfacing. However, the mechanism of dermal remodeling underlying this nonablative treatment remains unclear. METHODS: Five participants having skin phototype III-IV were recruited for intervention using a fractional picosecond 1064-nm Nd:YAG laser system equipped with a holographic diffractive beam-splitting optic. The laser-induced histopathological changes on human skin were examined in vivo using a harmonic generation microscopy (HGM), visualizing second harmonic generation (SHG), and third harmonic generation (THG) contrasts dichromatically. SHG refers for collagen distribution, while THG represents for epidermal components in the HGM signal. RESULTS: Histological hematoxylin and eosin staining and in vivo HGM imaging studies revealed the presence of epidermal vacuoles below the stratum granulosum along with keratinocyte degeneration or cytolysis. In addition to the epidermal vacuoles, HGM imaging exclusively demonstrated laser-induced shock wave propagation arranged as a THG-bright concentric pattern in the epidermis and loss of SHG signals in the papillary dermis immediately beneath the epidermal vacuoles. CONCLUSIONS: Alongside generating epidermal vacuoles, the fractional picosecond 1064-nm Nd:YAG laser induced collagen changes. These collagen changes may lead to dermal remodeling and neocollagenesis underlying the fractional picosecond laser treatment.

In vivo harmonic generation microscopy for monitoring the height of basal keratinocytes in solar lentigines after laser depigmentation treatment.

The development of solar lentigines (SLs) is related to chronic ultraviolet exposure-induced cell senescence. We have previously demonstrated that basal keratinocyte enlargement is a morphological hallmark of skin senescence correlated to the process of skin aging, while clinical studies on the long-term monitoring of the cellular morphological changes in SLs after laser treatment are lacking. In this study, we have developed the harmonic generation microscopy (HGM) for in vivo monitoring the height of basal keratinocytes (HBK) and had administered Q-switched ruby laser or picosecond 532-nm Nd:YAG laser treatment on each side of the face of 25 Asian patients with facial SLs, respectively. In vivo HGM imaging was conducted to longitudinally analyze HBK and the horizontal cell size (HCS). Before treatment, the HBK was significantly higher in the SLs lesional area than that in the adjacent normal region, whereas there was no significant difference in the HCS. After treatment, the lesional HBK remained significantly higher than normal skin regardless of the laser treatment used. Our study indicates that the basal keratinocytes remain abnormal after laser treatment and demonstrates the capability of in vivo HGM for longitudinal, quantitative monitoring of cell senescence and therapeutic effect in SLs.

Slide-free clinical imaging of melanin with absolute quantities using label-free third-harmonic-generation enhancement-ratio microscopy.

The capability to image the 3D distribution of melanin in human skin in vivo with absolute quantities and microscopic details will not only enable noninvasive histopathological diagnosis of melanin-related cutaneous disorders, but also make long term treatment assessment possible. In this paper, we demonstrate clinical in vivo imaging of the melanin distribution in human skin with absolute quantities on mass density and with microscopic details by using label-free third-harmonic-generation (THG) enhancement-ratio microscopy. As the dominant absorber in skin, melanin provides the strongest THG nonlinearity in human skin due to resonance enhancement. We show that the THG-enhancement-ratio (erTHG) parameter can be calibrated in vivo and can indicate the melanin mass density. With an unprecedented clinical imaging resolution, our study revealed erTHG-microscopy's unique capability for long-term treatment assessment and direct clinical observation of melanin's micro-distribution to shed light into the unknown pathway and regulation mechanism of melanosome transfer and translocation.