活體倍頻顯微術之光纖飛秒雷射

Abstract

活體組織切片為臨床醫學上疾病診斷的重要方法。病理醫師藉由觀察切片取 得的組織判斷患者疾病並幫助醫師做手術前的評估。切片組織通常會經由化學固 定、冷凍切片及染色的過程幫助病理醫師觀察細胞的型態以了解疾病的發展階段。 然而活體組織切片的樣品處理過程耗時且切片取樣會使病人感到疼痛，傷口造成 的流血也可能導致疾病細胞擴散的副作用。因此人們開始發展低侵入性且不須染 色的光學虛擬切片術。 現今的光學虛擬切片技術，包含了光學同調斷層掃描、共軛焦顯微術、多光 子顯微術及倍頻顯微術等。其中倍頻顯微術由於得到對比的方式為偵測光子與介 質耦合所產生的非線性光而非吸收光子所放出的螢光，所以與共軛焦顯微術及多 光子顯微術相比被認為是較低光傷害性的技術，另一方面由於非線性效應只會在 物鏡聚焦點產生的特性，可取得較光學同調斷層掃描術高的解析度的技術，此兩 種性質使得倍頻顯微術可以進行高解析度低傷害的活體影像的觀測。為了在活體 達到較深的穿透深度，倍頻顯微術通常會選擇對組織吸收及散射較低的1200 奈 米波長飛秒雷射光源作為激發光使用。現今能產生此波長範圍的雷射主要有兩種， 一為鈦藍寶石雷射再藉由光參震盪器調頻至1200 奈米、二為鉻貴橄欖石雷射直 接產生1200 奈米，此兩種雷射皆為固態雷射，固態雷射的輸出容易受溫度及濕 度影響。然而在臨床醫學的應用上，儀器的穩定性很重要，任何儀器的不穩定都 有可能直接影響到病人的生命安全，所以發展一套波長1200 奈米高穩定的飛秒 雷射系統是一個重要的技術課題。 在本研究中，我們利用雷射在光子晶體光纖中的孤子自頻移效應及準相位匹 配非線性晶體發展一套波長1160 奈米光纖飛秒雷射系統，希望可取代過去的固 態雷射光源，以提高活體倍頻顯微術的穩定性。

Biopsy is an important tool for clinical diagnosis. Pathologist examined the tissue removed from patients to determine whether or not diseases are present and help doctors do a pre-operative assessment. The removed sample will go through the processes of chemical fixation (or frozen occlusion), being sliced and staining to help pathologist recognize the cell morphology and observe the development of diseases. The procedure of biopsy is complicated and takes lots of time. When removing the tissues from patients, it also makes patients suffer and the bleeding may have side effects such as infection and spread of disease cells. For these reasons, the noninvasive and label-free virtual optical biopsy has been developed. Virtual optical biopsy includes optical coherence tomography, confocal microscopy, multiphoton fluorescence microscopy, and harmonic generation microscopy. Compared with other techniques, harmonic generation microscopy such as second harmonic generation microscopy and third harmonic generation microscopy provides higher penetration depth and sub-micron resolution and it leaves negeligible energy in specimens due to its characteristic of virtual-state-transition. Instead of collecting fluorescence produced by photon absorption, harmonic generation microscopy obtains image by detecting nonlinear signal from light-matter interaction. Therefore, it is considered as a less photo damage and less photo toxicity tool for a in vivo imaging technique. In order to achieve deeper penetration depth, it usually employs femtosecond laser sources around 1200 nm in which light-tissue interaction like photon absorption and scattering is greatly reduced. Conventional solid state femtosecond laser source like Ti : sapphire laser cascaded with optical parametric oscillator or Cr : forsterite solid state laser can provide the laser source around 1200 nm. Unfortunately, the performance of solid state laser is easily affected by temperature and humidity and it's not stable for medical use. In this thesis, we demonstrated a 7.5 MHz(&11.25MHz) repetition rate and 6.5 nJ pulse-energy fiber-based femtosecond laser source at 1160 nm. It was achieved by a soliton self-frequency shift in photonic crystal fiber and a second harmonic generation in a quasi-phase matching nonlinear crystal. We expect that such fiber-based laser system could replace the conventional solid state laser system to increase the stability of harmonic generation microscopy in clinical research.