時域聚焦之多光子激發顯微術

Temporal Focusing-Based Multiphoton Excitation Microscopy

本論文主要以時域聚焦所發展出的廣視域多光子激發顯微術應用於快速三維生醫影像與微細加工上，這技術目前影像大小為256×256像素的取像速度可達每秒100幀幅以上、側向解析度小於0.5 µm以及縱向解析度達3.5 µm，因此我們也藉由三種不同的技術來進一步提升成像的空間解析度。其中我們也利用數位微型反射鏡元件來替換系統中的繞射元件光柵，藉以達到可以同時保有時域聚焦以及任意圖案的照射。在空間解析度提升方面，首先藉由二階非線性結構式照明顯微術來提升系統的側向解析度至168 nm以及縱向解析度1.22 µm；另利用人工像散法可保有系統快速取像的能力來提升縱向解析度至約50 nm；最後藉由無波前感測式適應性光學系統修正樣品以及外界的干擾，進一步提升系統的照射效率以及影像品質。在生醫應用方面，除展現快速的三維生醫分子影像外，並完成任意三維圖形之蛋白質結構的微製作以及多光子誘發蝕除的應用。 A microscope based on temporal focusing offering widefi eld multiphoton excitation has been developed to provide fast three-dimensional(3D) biomedical imaging and microprocessing. This confi guration can produce multiphoton images at a frame rate greater than 100 Hz with a lateral spatial resolution of less than 0.5 m m and an axial resolution of approximately 3.5 m m. Also, three techniques have been adopted to promote the spatial resolution. Furthermore, by replacing the conventional diffraction grating with a digital micromirror device, the setup can provide temporal focusing and arbitrary pattern illumination simultaneously. In the spatial resolution improvement, the lateral and axial resolutions are enhanced to 168 nm and 1.22 m m by using a second order nonlinear structuredillumination microscopy. Then, the lateral resolution is further improved to 50 nm with the fast frame rate by integrating an astigmatism approach. Finally, a wavefront sensorless adaptive optics system was utilized to correct the disturbances induced from the sample and the environment. In the biomedical applications, the system provides fast 3D biomedical molecular imaging, but additionally, it also achieves freeform 3D protein microstructures and multiphoton-induced ablation.