螢光顯微術 (fluorescence microscopy)於生物醫學的應用中是一項很重要的工具，可以利用螢光分子標定目標物質，在螢光顯微鏡下直觀的看出此細胞結構或分子運動，相較於其他傳統顯微鏡，此技術高專一性的標記出待觀測分子，在觀察時能以高對比度進行觀測，而不易被其他分子影響。但在使用螢光顯微系統對具有相當厚度的生物樣品進行拍攝時，常因訊號穿透不同生物介質進而產生波前像差 (wavefront aberration)，這會對單分子定位(single molecule localization)技術造成相當大的影響，從而使螢光分子定位精度降低，單分子定位顯微影像的誤判也隨之增加。本論文主要將時域聚焦多光子激發單分子定位造影技術與波前修正系統結合，開發出可以使單分子定位技術愈加精準之系統。其中波前修正系統以Shack-Hartman波前感測器 (Shack-Hartman wavefront sensor)事先取出Zernike多項式中不同項次的模態，以可調變形鏡 (deformable mirror)進行波前修正；在使用螢光球樣品進行實驗時，可以發現在使用波前修正的情況下，得到的螢光訊號較無使用者強，且深度大之訊號點形狀經像差校正後，也較接近表面奈米球的形狀。最後利用適應性像散 (Adaptive Astigmatism，AA)代替圓柱透鏡，判斷不同奈米球在樣品中的深度資訊；於加入AA的實驗組中可以發現在不同深度時，像散的響應皆趨於一致，進而提升軸向判斷的能力。;Fluorescence microscopy is an important technique for the biomedical applications. The fluorescence microscope can be used to visualize the cellular structures and the molecular movements by labeling the target objects with the fluorophores, compared with other conventional microscopes. By the high-specific labeling to the target molecules, this technology has a capability of the high-contrast observation with the minimum disturbance signals from other molecules. However, when the fluorescence microscope is adopted to observe the thick bio-specimens, the fluorescence images suffer from the wavefront aberrations due to the fluorescence signal penetrating through different bio-media. The wavefront aberrations will have a big impact on the single-molecule localization for reducing the localization accuracy of fluorescent molecule and increasing the misjudgment of single-molecule localization microscopy imaging. In this thesis, the single-molecule localization microscopy system was developed by combined with the temporal focusing multiphoton excitation and the wavefront correction system to increase the localization accuracy. The wavefront correction system used the Shack-Hartman wavefront sensor to take the coefficients of different Zernike polynomials, and then countervailed the wavefront aberrations using the deformable mirror. After using the wavefront correction in the fluorescence-sphere measurement, the fluorescent signals of the fluorescence spheres were stronger than that without the wavefront correction. Moreover, the imaging shape of the deeper spheres were more identical to that of the spheres on the sample surface after the aberration correction. Finally, the adaptive astigmatism (AA) was adopted to instead of cylindrical lenses to extract the depth information of different spheres in the sample. In the experiment results with the AA, it can be found that the responses of the astigmatism tended to be the same at different depths, thereby improving the axial resolution.
