Optimization of Rapid Multiphoton Excitation Microscopy

• Main Authors: Tsung-YenChiang, 江宗晏
• Other Authors: Shean-Jen Chen
• Format: Others
• Language: en_US
• Published: 2017
• Online Access: http://ndltd.ncl.edu.tw/handle/xdezx7

碩士 === 國立成功大學 === 工程科學系 === 105 === Multiphoton excitation microscopy has been applied to many researches, including the field of biology and medicine. In biomedical science, there are lots of researches related to the variation of living cells. Therefore, a multiphoton excitation microscope with a high frame rate is needed to be built. By using a femtosecond laser which provide a high peak power within an ultrashort time interval, the nonlinear optical effect can be excited at the focal point. Moreover, with photon detectors, a galvanometer scanners, a resonance galvanometer scanner (RGS), and an electrically tunable lens, a three-dimensional (3D) rapid scanning multiphoton excitation microscope system can be realized. In this thesis, we use a galvanometer scanner and a resonance galvanometer scanner (Cambridge Technology Inc.) to achieve two-dimensional scan. The motion of resonance galvanometer scanner generates sinusoidal waves with 8 kHz resonant frequency. To compensate the nonlinear displacement causes the distortion of image, we adopt a pixel clock board (Cambridge Technology Inc.) to produce a compensated pixel trigger so that a 30 Hz frame rate imaging system can be achieved. After that, an electrically tunable lens was inserted to perform z-axis scanning to obtain a 3D image. In the light source part, a Ti:sapphire femtosecond laser manufactured by KMLabs was used. Having 95 MHz repetition rate can provide multiple excitation pluses within each pixel. A high speed and high gain transimpedance circuit is used as a current-to-voltage converter. Therefore, lower power can be utilized to get high signal-to-noise ratio (SNR) images so that the damage to biosamples can be reduced. Since the highest pixel rate is up to 15 MHz, we adapt a field-programmable gate array (FPGA) card with a high speed A/D module, which features 250 MS/s sampling rate as the resolution. Finally, the control program in LabVIEW is developed to handle the user interface and all the signals in the system.