時域聚焦多光子激發顯微術(temporal focusing multiphoton excitation microscopy，TFMPEM)能達到大面積的激發，提高幀速度，且保留了多光子的激發特性。TFMPEM中主要的元件有鈦藍寶石雷射再生放大器超快雷射(Ti:sapphire regenerative amplifier)為激發光源(重複率為10 kHz，脈衝能量在100 fs的脈衝寬度下高達400 μJ/pulse)，使用高靈敏度的電子倍增電荷耦合元件(electron multiplying charge-coupled device，EMCCD)來取像，在60 × 60 μm2的面積下，可得到幀速度高於100 Hz的大面積螢光影像。然影像的側向空間解析度(lateral spatial resolution)受限於光學繞射極限(diffraction limit)，被侷限在數百奈米以上，而軸向激發侷限(axial excitation confinement)受限於光學繞射極限與後焦平面(back-focal plane)光束能量分布問題，被限制在數個微米等級。為了突破影像側向空間解析度限制，採用了超高解析度非線性結構性照明顯微術來處理(super-resolution nonlinear structured-illumination microscopy，SRNSIM)，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，其側向解析度由420 nm減至230 nm，約提升1.82倍。
軸向激發侷限的突破，先以結構光繞射方向與不同光軸夾角方式實驗，再以不同空間頻率圖案進行，了解軸向激發侷限變化情形。因此設計出軸向激發侷限提升較多的結構照明來疊加和調變，可使系統軸向激發侷限由2.8 μm 提升到1.4 μm，約增進了2倍。最後，利用軸向激發侷限相差較大的結構照明打在樣品上做分析比較，驗證在軸向激發侷限提升較多的結構照明下，因所激發的軸向區域較小，所擷取到的雙光子螢光影像的結構圖案較清晰不模糊。

Temporal focusing multiphoton excitation microscopy (TFMPEM) has a larger excitation area. Moreover, it not only has high frame-rate capability but also holds the advantage of two photon excitation. In our lab-made TFMPEM system, the key components include a titanium-sapphire regenerative amplifier, a titanium-sapphire ultrafast oscillator as the seed beam of the amplifier, an upright optical microscope, an electron multiplying charge-coupled device (EMCCD) camera. The regenerative amplifier has a peak power of 400 μJ/pulse, with a pulse width of 90 fs and a repetition rate of 10 kHz. Based on the above configuration, the ability of axial resolution and the multiphoton excitation area is larger than 60 × 60 〖μm〗^2. Furthermore, the frame rate higher than 100 Hz can be achieved. Due to the spatial resolution restricted by the diffraction limit, the lateral resolution is limited in sub-nanometers and the axial resolution is limited in submicrons. Dealing with the diffraction limit, the super resolution linear/nonlinear structured illumination microscopy (SR-SIM/NSIM) has been adopted.
In this thesis, first, we tried different diffraction angles combine with different spatial frequency sinusoidal patterns to estimate the characteristic of axial resolution. Due to the observation, we designed structured patterns with different orientation and changed spatial frequency. In the result of our experiments, we can design a pattern to achieved the axial resolution from 2.8 μm to 1.4 μm, it increased two times effect than before. Finally, we demonstrated that the different axial confined enhancement will affect the contract of images when we focused on the same depth in a temporal focusing setup.

[1] D. Oron, E. Tal, and Y. Silberberg, “Scanningless depth-resolved microscopy,” Opt. Express 13, 1468-1476 (2005).
[2] G. Zhu, J. van Howe, M. Durst, W. Zipfel, and C. Xu, “Simultaneous spatial and temporal focusing of femtosecond pulses,” Opt. Express 13, 2153-2159 (2005).
[3] A. Vaziri, J. Tang, H. Shroff, and C. V. Shank, “Multilayer three-dimensional super resolution imaging of thick biological samples,” Proc. Natl. Acad. Sci. U.S.A. 105, 20221-20226 (2008).
[4] E. Papagiakoumou, F. Anselmi, A. Bègue, V. de Sars, J. Glückstad, E. Y. Isacoff, and V. Emiliani, “Scanless two-photon excitation of channelrhodopsin-2,” Nat. Methods 7, 848-854 (2010).
[5] W. Denk, J. H. Strickler, and W. W. Webb, “Two-photon laser scanning fluorescence microscopy,” Science 248, 73-76 (1990).
[6] F. Helmchen and W. Denk, “Deep tissue two-photon microscopy,” Nat. Methods 2, 932-940 (2005).
[7] M. G. Gustafsson, D. A. Agard, and J. W. Sedat, “Doubling the lateral resolution of wide-field fluorescence microscopy using structured illumination,” Proc. SPIE 3919, 141-150 (2000).
[8] M. Goepper-Mayer, “Ober Elementaraktemit zwei Quantensprungen,” Annalen der Physik 9, 273-294 (1931).
[9] M. G. Gustafsson, “Nonlinear structure-illumination microscopy: Wide-field fluorescence imaging with theoretically unlimited resolution,” Proc. Natl. Acad. Sci. U.S.A. 102, 13081-13086 (2005).
[10] L.-C. Cheng, C.-H. Lien, Y. D. Sie, Y. Y. Hu, C.-Y. Lin, F.-C. Chien, C. Xu, C. Y. Dong, and S.-J. Chen “Nonlinear structured-illumination enhanced temporal focusing multiphoton excitation microscopy with a digital micromirror device,” Biomed. Opt. Express 5, 2526-2536 (2014).
[11] M. G. Gustafsson, L. Shao, P. M. Carlton, C. J. Wang, I. N. Golubovskaya, W. Z. Cande, D. A. Agard, and J. W. Sedat, “Three-dimensional resolution doubling in wide-field fluorescence microscopy by structured illumination,” Biophys. J. 94, 4957-4970 (2008).
[12] K. Isobe, T. Takeda, K. Mochizuki, Q. Song, A. Suda, F. Kannari, H. Kawano, A. Kumagai, A. Miyawaki, and K. Midorikawa, “Enhancement of lateral resolution and optical sectioning capability of two-photon fluorescence microscopy by combining temporal-focusing with structured illumination,” Biomed. Opt. Express 4, 2396-2410 (2013).
[13] W. Lukosz and M. Marchand, “Optischen Abbildung unter Überschreitung der beugungsbedingten Auflösungsgrenze,” Opt. Acta 10, 241-255 (1963).
[14] M. A. A. Neil, R. Juˇskaitis, and T. Wilson, “Method of obtaining optical sectioning by using structured light in a conventional microscope,” Opt. Lett. 22, 1905-1907 (1997).
[15] M. G. Gustafsson, “Surpassing the lateral resolution limit by a factor of two using structured illumination microscopy,” J Microsc. 195, 10-16 (2000).
[16] S. T. Hess, T. P. Girirajan, and M. D. Mason, “Ultra-high resolution imaging by fluorescence photoactivation localization microscopy,” Biophys. J. 91, 4258-4272 (2006).
[17] M. J. Rust, M. Bates, and X. Zhuang, “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nat. Methods 3, 793-796 (2006).
[18] T. Dertinger, R. Colyer, G. Iyer, S. Weiss, and J. Enderlein, “Fast, background-free, 3D super-resolution optical fluctuation imaging (SOFI),” Proc. Natl. Acad. Sci. U.S.A. 106, 22287-22292 (2009).
[19] D. Kim, Ultrafast Optical Pulse Manipulation in Three Dimensional-Resolved Microscopic Imaging and Microfabrication, PH.D. Thesis, MIT (2009).
[20] J. W. Goodman, Introduction to Fourier Optics, Roberts & Company (2005).
[21] 鄭力中，廣視域多光子激發顯微術之開發與應用，國立成功大學光電科學與工程研究所碩士論文 (2010)。
[22] J. Guild, C. Xu, and W. Webb, “Measurement of group delay dispersion of high numerical aperture objective lenses using two-photon excited fluorescence,” App. Opt. 36, 397-401 (1997).
[23] M. Durst, G. Zhu, and C. Xu, “Simultaneous spatial and temporal focusing for axial scanning,” Opt. Express 14, 12243-12254 (2006).
[24] S. Santos, K. K. Chu, D. Lim, N. Bozinovic, T. N. Ford, C. Hourtoule, A. C. Bartoo, S. K. Singh, and J. Mertz, “Optically sectioned fluorescence endomicroscopy with hybrid-illumination imaging through a flexible fiber bundle,” J. Biomed. Opt. 14, 030502 (2009).