Improvements of Temporal Focusing-based Multiphoton Excitation Microscopy by Using Structured Illumination

• Main Authors: Chen-HanLin, 林承翰
• Other Authors: Shean-Jen Chen
• Format: Others
• Language: zh-TW
• Published: 2016
• Online Access: http://ndltd.ncl.edu.tw/handle/95128688423262023711

碩士 === 國立成功大學 === 工程科學系 === 104 === 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.