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Traditional biopsy requires the removal, fixation, and staining of tissues from the human body. Its procedure is invasive and painful. Therefore, a novel method of optical biopsy is desired which can perform in vivo examination and is noninvasive, highly penetrative, with no energy deposition and damage, without invasive pharmaceutical injection, and with three-dimensional (3D) imaging capability and sub-micron spatial resolution. Two-photon fluorescence microscopy (TPFM) is previously applied for biopsy of skin due to its high lateral resolution, low out-of-focus damage, and intrinsic 3D section capability. However, for future clinical applications without surgery, current 700-850 nm based laser scanning technology still presents several limitations including low penetration depth, in-focus cell damages, multi-photon phototoxicity due to high optical intensity in the 800 nm wavelength region, and toxicity if exogenous fluorescence markers were required. Here we demonstrate a novel noninvasive optical biopsy method called harmonics optical biopsy (HOB), which combines both second harmonic generation imaging and third harmonic generation imaging. Due to virtual transition nature of harmonic generations and based on light sources with an optical wavelength located around the biological penetration window (~1300nm), our HOB can serve as a truly non-invasive biopsy tool with sub-micron three-dimensional spatial resolution without any energy deposition and exogenous contrast agents. From preliminary experiment result, our HOB can reconstruct 3D cellular and subcellular images from skin surface through dermis. Besides, by utilizing backward propagating detection geometry, we will show that this technique is ideal for non-invasive clinical biopsy of human skin diseases and even useful for the early diagnosis of skin cancer symptom such as the angiogenesis.
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