雙光子螢光顯微術比起傳統的廣域顯微鏡有著高縱向解析度的特點和高穿透深度的優點，故廣泛地被應用在生物醫學研究與臨床實驗。然而雙光子顯微鏡的空間解析度受限於光學繞射極限，低於繞射極限結構的樣本將無法被解析。為了在生物厚組織中也能取得優秀於傳統雙光子顯微術解析度的影像，本研究將結合結構照明與雙光子顯微術的概念開發一套超解析度影像系統，稱之為雙光子掃描結構照明顯微術(Two-photon scanning structured illumination microscopy, TPS-SIM)。 在此系統中，強聚焦雷射光激發樣本並產生雙光子螢光訊號，在雷射掃描過程中藉由二維相機收集每個激發光點的訊號並組成影像，若控制其光點掃描路徑使激發光強度分布類似方波，在此結構照明下將可獲得傳統影像系統無法擁有的高空間頻率資訊。多筆具有結構照明的影像資料經過演算重建後，將可以產生更高解析度的影像。在本論文中將會詳述系統成像之理論模型及影像重建之演算法，並討論條紋週期大小、佔空比(Duty cycle)、等效明晰度與影像訊雜比對解析度的影響。藉由測量螢光奈米小球的螢光強度分布，可知TPS-SIM在橫向上最大解析度提升倍率為1.42倍。結合雙光子系統所具有的光學切片能力，本系統可在蘇木素•伊紅染色的動物組織切片與老鼠皮膚中取得三維影像，除橫向解析度的提升外，其縱向解析度最大可提升至1.67倍。 ;Compared with conventional wide-field microscopy, two-photon microscopy (TPM) has advantages of inherent axial resolution and high penetration. Therefore, TPM has been widely applied to bio-medical and clinical researches. However, the spatial resolution of TPM is restricted by the optical diffraction limit so structures smaller than the limit can’t be resolved. To improve the resolution of TPM image in depth tissue, this research will integrate the concept of structured illumination and TPM to develop an imaging system called two-photon scanning structured illumination microscopy (TPS-SIM). In this system, laser beam is tightly focused onto sample to excite two-photon fluorescence signals. The excited signals are imaged and integrated by 2D camera point by point to form an image. During the scanning procedure, the path of the excitation spot is modulated to form an effective structured illumination with a square-wave intensity distribution. Under this structure illumination, higher spatial frequency out of the reach of the conventional wide field microscopy can thus be obtained. An image with improved resolution can be reconstructed though multiple patterned images with different phases. In this research, the theory of the image formation and the image reconstruction algorithm will be clearly introduced. The effects that the period, duty cycle, effective modulation depth of pattern and SNR (signal and noise ratio) may have on the resolution improvement will be discussed. By measuring the fluorescence intensity distribution of the nanoparticles, the maximum resolution improvement ratio of TPS-SIM is around 1.42-fold. Combined the optical sectioning ability of two-photon excitation, 3D images can be obtained in H&E stained sectioned bio-tissues and fluorescence stained whole-mounted mouse skin. In addition to improvement in lateral resolution, the maximum improvement ratio in axial is around 1.67-fold.
