本論文開發出具多光子時域聚焦照明之光場顯微鏡(light-field microscope with multiphoton temporal focusing illumination)，其具有光場顯微術的特性，可利用單一張影像還原出一定體積的影像，且藉由多光子時域聚焦照明，可以做到多光子激發，得到較深的穿隧深度、對樣品較少的破壞、以及可選定範圍之縱向侷限激發的特性，進而得到選取觀測範圍，降低背景雜訊的優點。綜合以上特性，該系統擁有高速的影像紀錄能力及相較更為低的雜訊，適合用來觀測快速的生物體影像。我們利用800 nm的超快雷射作為激發雷射，使用40倍數值孔徑為1的水鏡作為物鏡成像，經過2.5倍的放大透鏡組到間距為125 μm的方形微透鏡陣列上，在藉由1:1.5的延伸透鏡轉接至Electron-Multiplying CCD (EMCCD)以覆蓋更多像素。依據此配置，理論上可以在大約上下2 μm的地方得到0.8 μm的解析度，並利用時域聚焦的特性，在物鏡的後焦平面處放置一個可調式的光圈，調整覆蓋於後焦平面的面積，進而得到約30 μm的縱向激發長度，搭配約100 μm ⨯ 100 μm的橫向激發面積，達到選擇激發體積的目的。由於使用的還原演算法3D反卷積的計算是屬於事後的演算，因此系統的體成像數取決於EMCCD的取影像速度，因此適合用於動態的生物體影像。以2 μm的螢光球來說，單一張曝光時間使用了0.05秒，也就是我們的體積影像速率可以達到20 Hz，以幀數來來換算就是20 Hz⨯20層的每秒400張影像。

Light field, known as its fast slicing image, is suitable for capturing dynamic sample. With single shot light field, it can get the whole information of the volume. That is, we can keep the original frame rate of the system, and reconstruct the three-dimensional (3D) image. In light field microscope, we can use high numerical aperture objective to obtain higher angular frequency. We choose the microlens array whose f-number can match our objective; therefore, the subimage for a single lenslet will not cover to each other. Utilizing 3D deconvolution algorithm to deblur the out-of-focusing part. Light field microscope illuminates the whole volume of sample; nevertheless, whole volume excitation may cause larger damage to biological tissue. In addition, the reconstruct criterion of light field microscope is limited. The fluorescent out of range may become background noise and degrade the image quality. In this research, we implement light field microscope to temporal focusing. Then, we utilize a slit on the back focal plane of the objective to control the axial confinement excitation area, and let the excited volume match the range we can reconstruct by light field algorithm. As a result, we demonstrate the light field microscope with temporal focusing can reconstruct 3D image, and the lateral resolution is approaching to theoretical value. Moreover, with better signal-to-noise ratio and less damage to tissue, the system can observe biological samples and reconstruct 3D image.

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