利 用 同 時 空 間 與 時 間 聚 焦 (simultaneously spatial and temporal focusing)技術的廣視域多光子激發螢光顯微技術(widefield multiphoton excited fluorescence microscopy)有機會快速提供活體生物組織的三維(three-dimensional，3D)多光子螢光影像。不同於一般傳統式的多光子螢光顯微術需要逐點掃描，廣視域多光子顯微技術僅需要掃描一個軸向，也就是z 軸，藉此去建構一個3D 影像。本論文利用Ti:sapphire 飛秒雷射振盪器(femtosecond laser oscillator)當作飛秒雷射放大器(femtosecond laser amplifier)的根本光源(seeding source)，輸出的120 fs 脈衝雷射其重覆率(repetition rate)為10 kHz，也將原先的脈衝雷射之能量提升至4.2 瓦，而這樣的瞬間極大能量足夠去激發大於100 x 100 μm2 面積的多光子螢光信號。此外，軸向的解析度可以到達5 μm以下，再利用冷卻式電子倍增感光耦合器(electron multiplying charge couple device，EMCCD)照相機來擷取螢光影像信號，其幀速率(frame rate)依照不同螢光團分子的放出效率而有不同的值，其最高可達數百Hz。因此具有此飛秒雷射放大器與低溫EMCCD 照相機的協助，此廣域多光子顯微技術可以提供非常快速擷取活體生物影像，例如可以即時動態觀測腦部神經細胞的活動以及肝臟的代謝等。目前此系統的縱向解析度可達將近2.5 μm，也利用量測微米螢光球來推估其側向解析度達500 nm 以下。另外，成功地針對腦神經細胞組織進行多光子螢光3D 影像重建，目前使用EMCCD 全範 圍1000 x 1000 的像素其幀速度最快可達20 Hz。

A widefield multiphoton excited fluorescence microscopy based on simultaneously spatial and temporal focusing technique has potential for providing fast three-dimensional (3D) multiphoton excited fluorescence images for bio-tissues in vivo. Unlike conventional multiphoton microscopy based on pixel by pixel scanning technique, the widefield multiphoton microscopy detects sequential 2D images via axial scanning, i.e. z-axis, and converts them to construct a complete 3D image. By using a Ti:sapphire femtosecond laser oscillator as a seeding source to feed a femtosecond laser amplifier, an instantly extreme power at 120 fs pulse width and 10 kHz repetition rate from 4.2 W average power can achieve multiphoton excited fluorescence imaging with the excitation area over 100 x 100 μm2. The fluorescence image is detected by a TE-cooled electron multiplying charge couple device (EMCCD) camera whose frame rate can be over 100 Hz according to the emission efficiency of fluorophores. With the help of the femtosecond laser amplifier and the EMCCD camera, the widefield multiphoton excited fluorescence microscopy can provide very fast imaging for bio-tissues in vivo such as monitoring brain neuron activity and liver metabolism. Currently, the axial resolution of the system is about 2.5 μm and the lateral resolution is better than 500 nm based on the testing of fluorescence micro-beads. Furthermore, the 3D images of neuron cell tissues have been successfully achieved with a frame rate of 20 Hz at the full range of 1000 x 1000 pixels.

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