臨床倍頻顯微術：皮膚之光學虛擬切片

Abstract

倍頻顯微術結合了二倍頻及三倍頻顯微術，並以一鉻貴橄欖石為激發光源，因其同時擁有非侵入性、高解析度及高穿透度等優點，在醫學應用上，為一可用來進行非侵入性虛擬光學切片的新技術。在皮膚裡，三倍頻的信號可來自於物質的非同質性、細胞胞器、脂質、血紅素以及黑色素；而二倍頻信號的主要來源則是第一型的膠原蛋白，基於這些豐富多樣的信號來源，倍頻顯微術可被用來觀察細胞型態或是膠原蛋白纖維結構上的病變，此技術在未來很適合應用於皮膚疾病的臨床診斷。 在本論文中，我們用反射式倍頻顯微術〈背向收集訊號〉來活體外觀察手術過程中所切割下來的皮膚樣本，包括病變及正常的皮膚；另外，我們也第一次使用倍頻顯微術在活體內觀察21位健康受試者前臂的皮膚。在手術皮膚樣本的研究中，三倍頻影像被證實與組織切片有相當好的一致性，且皮膚裡多樣的三倍頻信號來源也可經由三倍頻影像與組織染色切片的比對來一一辨認，此研究不但顯示了倍頻顯微術在診斷各種不同皮膚疾病及分辨良性與惡性病變上的能力，更進一步指出三倍頻顯微影像可以用來進行黑色素的分子影像，及早期診斷與黑色素相關的皮膚病變。除了細胞型態上的資訊，經由在三倍頻影像上進行細胞學分析的結果得知，三倍頻影像還可提供更多有醫學診斷價值的細胞學資訊。 另一方面，在活體內的研究中，我們觀察了21位不同膚色及不同年齡的健康受試者的前臂皮膚，在觀察過程中，除取得倍頻影像外還同時進行了本系統的傷害評估，為減低取影像時因呼吸及心跳所導致的振動，我們設計並使用了用來固定手臂皮膚的設備，如注射器幫浦轉接器及真空幫浦吸盤等。取影像時，在雷射通過一數值孔徑為1.2的物鏡後所量得的平均雷射功率為90mW，經過三十分鐘連續的雷射曝射，甚至在膚色最深的受試者皮膚上也沒有觀察到任何的傷害，由此傷害評估的結果可確認倍頻顯微術在人體實驗上的非侵入性及安全性。相較於活體外的倍頻影像只能取得靜態的資訊，皮膚中的寶貴的動態資訊如血流亦可即時地被活體內的倍頻影像記錄下來，即使有振動的影響，在整個300um的穿透深度內不論是二倍頻還是三倍頻顯微術都能保有次微米的橫向解析度，且相較於過去在福馬林固定的皮膚樣本裡所量得解析度隨著影像深度的變化，活體皮膚內解析度隨著深度衰退的程度較為輕微。基於三倍頻在皮膚表皮層有很強的信號且擁有很高的解析度，活體內取得的三倍頻影象亦可被用來進行細胞學的分析，且分析所得的結果和活體外所取得的資訊一致，展現出其結果在疾病診斷上的重要性。 在活體內觀察不同膚色的皮膚的研究中，經由數值分析及與染熱切片的比對，我們進一步確認了黑色素在三倍頻信號增強及衰減所扮演的主要角色，藉著由黑色素引起的三倍頻信號的增強，三倍頻顯微術可提供在黑色素分子影像及追蹤黑色素相關皮膚病變方面的能力。在皮膚老化的活體內的初期研究中，包含了內在及外在〈光〉老化的探討，經由三倍頻顯微術，我們可以觀察到內在老化所導致的細胞擴大，及光老化所引起的黑色素含量增加，而藉由二倍頻顯微術則可觀察到由內在老化及光老化所導致的各種膠原蛋白纖維結構的變化。

With the ability to achieve noninvasiveness, high resolution, and high penetrability at the same time, Cr:forsterite-based harmonic generation microscopy (HGM), combining both second and third harmonic generation (SHG and THG) modalities, is a new paradigm for in vivo non-invasive virtual optical biopsy. Based on various THG contrasts arising from optical inhomogeneity, cytoplasm organelles, lipids, hemoglobin, and melanin, and SHG contrast arising from type I collagen, HGM shows strong capability for revealing the pathological changes of cellular and collagenous morphology and is a promising tool for future noninvasive diagnosis of skin diseases. In this thesis, ex vivo epi-HGM (HGM with backward-collection geometry) imaging has been performed on freshly excised normal and lesional human skin specimens, and for the first time, in vivo epi-HGM imaging has been demonstrated on 21 health volunteers’ forearm skin. In the ex vivo studies, epi-THG imaging is shown to possess strong histology consistence and various THG contrasts in human skin have been identified through histology comparisons. The ex vivo studies not only demonstrate the ability of epi-HGM to histopathologically distinguish amongst different skin diseases and between benign and malignant lesions; but also indicate the unique capability of THG imaging for molecular-imaging of melanin and early diagnosing the melanocytic lesions. In addition to revealing the morphological information, the results of the epi-THG-based cytological analysis further show that epi-THG can also provide the cytological information for skin disease diagnosis. In the in vivo studies, health forearm skin of volunteers with different skin colors and different ages has been investigated along with damage evaluation. To reduce the vibrations due to breathing and heart beating, devices like syringe-pump adapter and vacuum-pump sucker have been designed and used for sample stabilization. During 30 minutes of continuous laser excitation with an average power of 90 mW after a NA 1.2 objective, no photodamage was reported even in the heavily-pigmented skin and the noninvasiveness and safety of in vivo epi-HGM biopsy are confirmed. In contrast to the ex vivo imaging, in vivo epi-HGM imaging demonstrates the capability to record valuable dynamic information like blood flow in the dermis. Even with vibrations due to breathing and heart beating, sub-micron lateral resolution is possessed by both epi-SHG and epi-THG modalities throughout a ~ 300um imaging depth and less signal degradation is found in live human skin than in fixed human skin. Based on the strong THG contrasts in epidermis and high spatial resolution of the in vivo epi-THG imaging, the epi-THG-based cytological analysis can also be performed in vivo and the in vivo results shows strong consistence with ex vivo results and diagnostic significance. In the in vivo epi-HGM imaging of differently-pigmented skin, the primary role of melanin in THG enhancement and attenuation has been further confirmed through both statistic analyses and histology comparisons. With the melanin-induced THG enhancement, epi-THG modality demonstrates strong capability for molecular-imaging of melanin and screening melanocytic skin lesions. In the preliminary in vivo study of skin aging, both the intrinsic skin aging and photo-aging have been investigated. Epi-THG modality is shown to be able to reveal the enlargement of keratinocytes in intrinsic-aged skin and increasing content of melanin in photo-aged skin; while epi-SHG modality is shown to have the ability to reveal the changes of collagenous structures in dermis due to both intrinsic aging and photo-aging.