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|Title: ||雙光子掃描結構照明顯微術;Two-photon Scanning Structured Illumination Microscopy|
|Keywords: ||非線性顯微術;螢光顯微術;超解析顯微術;結構照明;切片影像;Nonlinear microscopy;Fluorescence microscopy;Super-resolution microscopy;Structured illumination;Sectioning image|
|Issue Date: ||2016-10-13 13:17:18 (UTC+8)|
|Abstract: ||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)。|
;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.
|Appears in Collections:||[光電科學研究所] 博碩士論文|
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