In vivo harmonic generation biopsy of human skin

Abstract

The ability to in vivo image deep tissues noninvasively with a high resolution is strongly required for optical virtual biopsy. Higher harmonic generation microscopy, combined with second- and third-harmonic generation microscopies, is applied to 17 Asian volunteers' forearm skin. After continuous observation for 30 min, no visible damage was found. Our study proves that harmonic generation biopsy (HGB) is able to satisfy the safety requirement and to provide high penetrability (approximately 300 microm) and submicron resolution all at the same time and is a promising tool for future virtual biopsy of skin diseases. In contrast to a previous study on fixed human skin specimens, a much improved penetrability and much reduced resolution-degradation versus depth are found in this in vivo examination.

Differentiating intratumoral melanocytes from Langerhans cells in nonmelanocytic pigmented skin tumorsin vivoby label-free third-harmonic generation microscopy

“…[27][28][29][30][31][32][33][34] Based on the principle of third-order nonlinear processes, the melanin-enhancement nature of label-free third-harmonic generation (THG) in HGM has been confirmed in the previous study. 28 We have also demonstrated that HGM achieved high diagnostic accuracy in nonmelanoma pigmented skin lesions such as BCC, SK, and melanocytic nevus.…”

“…In the histopathological examinations, no evidence of photodamage, such as coagulation necrosis, on the illuminated specimens was identified. 27,28,31-34…”

“…27,28,[31][32][33][34] The irregular THG-bright cells with dark nuclei and multiple cellular processes longer than the cell bodies were found mainly in the basal layer of the epidermis and inside certain tumors through THG microscopic imaging without any staining to the skin tissues. Due to the strong THG signals, enlarged cell volumes, and irregular shapes with several dendritic processes, the dendritic cells can be morphologically distinguished from the surrounding small, polygonal basal keratinocytes (Fig.…”

Third harmonic generation microscopy of cells and tissue organization

“…THG has the potential to complement these existing imaging approaches. The applicability of THG in optical biopsies has been demonstrated in hamster and human oral mucosa, as well as human skin and adipose tissue (Chen et al, 2009;Tai et al, 2005Tai et al, , 2006Tsai et al, 2011Tsai et al, , 2013. Furthermore, the image contrast generated using THG is suitable for discriminating melanoma and basal cell carcinoma lesions from normal skin and benign nevi .…”

In vivo imaging of cell nuclei by photoacoustic microscopy without staining

“…The high image contrast results from a difference between strong light absorption of nucleic acids and weak absorption of protein, which reaches the maximum at a wavelength of 250 nm. In addition to UV-PAM, several other modern optical microscopy technologies have been explored for in vivo imaging of unstained cell nuclei, such as reflectance confocal microscopy [8][9][10][11], multiphoton microscopy [12][13][14], and third-harmonic generation microscopy [15,16], However, reflectance confocal microscopy is difficult to provide specific image contrast for nuclei [8], multiphoton microscopy produces in vivo images of cell nuclei with negative contrast [13], and third harmonic generation microscopy generates nuclear images with low contrast [15].…”