微型化微機電鏡掃描之背向三倍頻光纖顯微鏡

Abstract

在這篇論文中，我們提出並已建立了第一套微型化微機電鏡掃描之背向倍頻光纖顯微鏡，包含了背向二倍頻及背向三倍頻顯微鏡。由於產生倍頻時能量守恆的特性，訊號產生的過程中不會有多餘能量累積在樣本。因此，倍頻顯微術提供了最低侵入式的顯微影像。另外，倍頻訊號對入射光的強度有很高度的敏感性，只有在聚焦焦點的位置才會有足夠強的倍頻訊號，這樣的特性提供了倍頻顯微術非常好的光學切片能力。利用倍頻顯微術並結合高數值孔徑的物鏡，不需要外加的共軛焦孔隙就可以達到次微米級的空間解析度。 利用倍頻顯微術觀察生物樣本，二倍頻訊號主要產生於生物體內整齊排列的奈米級構造，而三倍頻則是產生於介面不連續處提供完整的細胞型態資訊。因此，利用此技術可以呈現出樣本中細胞級和次細胞級的微結構，結合上述優點，此技術非常適合用來做光學虛擬切片。 在發展倍頻顯微術之前，必須先尋找適合的激發光源。另外，不論在倍頻顯微術或是光纖非線性現象的研究上，高功率的光源是必須的。由於飛秒鉻貴橄欖石(Cr:forsterite)雷射做為激發光源有著較深的穿透深度及較少的光破壞，於是此研究的第一個課題是建立一套高功率的鉻貴橄欖石雷射。藉由比較使用不同鉻貴橄欖石晶體的雷射輸出效能，我們建立的單一共振腔飛秒雷射可達到破紀錄的1.3瓦平均功率。另外，高穩定性的輸出表現使得這台雷射可以直接用來做倍頻影像而不需要額外的飽和吸收機制。接下來，為了進一步手持式系統的開發，必須建立光纖傳輸的光源以增加系統使用的靈活性。我們利用大模態光子晶體光纖(large-mode-area photonic crystal fiber)來降低在光纖中傳光時造成的非線性效應並使用此光纖來傳輸飛秒脈衝光源。 系統微小化及高畫面重覆率是接下來的研究課題。高畫面重覆率的需求主要是來自於受試者端很難避免的震動或移動。利用二維掃描之微機電系統掃瞄鏡、一對自行設計的透鏡組及商用物鏡，我們成功的建立了一套微小化且利用光纖傳輸的倍頻顯微鏡。結合場域可程式化閘陣列(FPGA)，系統的畫面更新率可達到每秒30張。從一微米直徑的螢光小球溶液觀測中，此系統可以達到0.4微米的直接背向三倍頻解析度。在活體人體皮膚的觀察方面，我們可清楚觀察到皮膚表皮層的細胞結構及真皮層的膠原纖維。同時利用此系統，真皮層中微血管及血流的動態觀察亦可被實現。

In this thesis, the first miniaturized MEMS-based epi-harmonic-generations fiber-microscope (HGM) was demonstrated, including both epi-second-harmonic-generation (epi-SHG) microscope and epi-third-harmonic-generation (epi-THG) microscope. Because of the nature of energy conversation of harmonic generation (HG) processes, HG leaves no energy deposition to the interact specimen. Therefore, it is the least invasive optical microscopic technique. In addition, since the generated HG signals are sensitive to the incident intensity, sufficient signals can be found only near the focal region and consequently HGM provides the intrinsic optical sectioning capability. Combining with a high numerical aperture (NA) objective, the sub-µm spatial resolution can be achieved without the need of confocal pinhole. SHG signals are mainly generated from the highly in-order nano-structures in the biological specimens and THG is sensitive to interfaces which provide the information of cell morphology. Thus, the cellular and sub-cellular structure of the specimen can be visualized using HGM and it is a desirable tool for optical virtual biopsy. In regard of the development of HGM, the suitable excitation source should be found. A Cr:forsterite laser was chosen as the excitation source because both high penetration depth and the reduced photodamage could be achieved using this laser. Besides, the high average power of the laser is necessary for not only HG imaging but also the studies of fiber nonlinearities. Thus, in this work, a high power Cr:forsterite laser was built by comparing the performance of using different Cr:forsterite crystals. A world-record-high average power, 1.3W, can be achieved in a single resonator. This Kerr-lens-mode-locking laser was stable enough for imaging without the need of other saturation absorber. For further hand-held applications, the imaging system should be fiber-based to increase the flexibility of the system. A large-mode-area (LMA) photonic crystal fiber (PCF) was chosen to reduce the nonlinear effect in the fiber and the fiber was used to deliver the femtosecond pulse. The system miniaturization is another key issue and high frame-rate imaging is also required because it is hard to preventing the vibrations and the movements of the volunteer. With a 2D-scanning micro-electro-mechanical system (MEMS) mirror, a pair of well-designed relay lenses, and a commercial objective, we successfully built a miniaturized fiber-based HGM. With the aid of a Field Programmable Gate Array (FPGA) card, imaging acquisition with a video rate (30Hz) was obtained. A 0.4 µm spatial resolution of direct epi-THG imaging was also achieved and examined by observing 1µm-diameter fluorescent beads. In regard to in vivo observation of human skin, the cellular structures in the epidermis and the collagen fiber in the dermis could be observed clearly in the sectioned images. The capillary and the red blood flow in the dermis were also visualized.