時域聚焦多光子激發顯微術(temporal focusing multiphoton excitation microscopy，TFMPEM)主要以鈦藍寶石雷射再生放大器超快雷射(Ti:sapphire regenerative amplifier)為激發光源(其重複率為10 kHz且脈衝能量在100 fs的脈衝寬度下高達400 μJ/pulse)，加上繞射光柵、準值透鏡以及物鏡所構成的4f架構，以達到具縱向解析能力與面多光子激發，最後以高靈敏度的電子倍增電荷耦合元件(electron multiplying charge-coupled device，EMCCD)來取像，可得到幀速度高於100 Hz的大面積螢光影像。另外，因其空間解析度受限於光學繞射極限(diffraction limit)，使得側向解析度大約侷限在數百奈米，而縱向解析度約在數個微米等級。為了突破此限制，線性/非線性結構性照明顯微術(linear/nonlinear structured-illumination microscopy，SIM/NSIM)可採用。
論文中利用數位微型反射元件(digital micromirror device，DMD)取代傳統光柵，同時作為繞射元件以及產生結構圖案，並探討激發圖案對TFMPEM之縱向解析度的影響。實驗中發現，若使用弦波結構圖形激發時，縱向解析度會隨著結構圖形空間頻率變差，從2.5 μm逐漸上升到3.2 μm，但若結構圖形之空間頻率高至系統無法接收時，縱向解析度又會變為原來的2.5 μm。故可以得知特殊的照射圖形在經過傅立葉轉換之後，若能夠控制物鏡後焦平面雷射的頻率分布，將有機會調變縱向解析度。另外，藉由NSIM提升空間解析度，其側向解析度由400 nm減至227 nm (提升1.76倍)，並進一步以小鼠腦皮脂切片厚度定義我們能穿透的深度，藉由NSIM減少背景雜訊與散射光的優點，使目前可以觀察深度達到60 μm腦皮脂厚度下的200 nm螢光球影像。

In this thesis, a developed temporal focusing multiphoton excitation microscope (TFMPEM) in our laboratory utilized a Ti:sapphire regenerative amplifier as the excitation source and a diffraction grating, a collimating lens, and an objective lens to construct a 4f setup. Then, a high sensitivity electron multiplying charge-coupled device (EMCCD) to receive was used for imaging. Based on the above configuration, the ability of axial resolution and the multiphoton excitation area larger than 40×40 〖μm〗^2 at a frame rate faster than 100 Hz can be achieved. Due to the spatial resolution restricted by the diffraction limit, the lateral resolution is limited in hundreds of nanometers and the axial resolution is limited in few microns. To overcome this limitation, linear/nonlinear structured illumination microscopy (SIM/NSIM) has been adopted.
We use a digital micromirror device (DMD) to replace a traditional optical grating. As sinusoidal illumination patterns used to perform TFMPEM, the axial resolution was worse from 2.5 μm to 3.2 μm when the spatial frequency of the structured illumination pattern became higher. But if the spatial frequency of the structured structure illumination pattern was higher than the system’s cutoff spatial frequency, the axial resolution will recover to 2.5 μm. Also, the NSIM can enhance the lateral resolution from 400 nm to 227 nm (1.76 folds). Furthermore, mice brain cortex slices were utilized to test the penetration depth of the TFMPEM. Currently, the 200 nm fluorescence beads images under a 60 μm thick mouse brain cortex slice can be improved.

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