開發「鎖相放大倍頻顯微技術」於肌纖維收縮之觀測

Abstract

掃描式光學影像顯微技術依照掃描方式可以分為雷射光束掃描與樣品移動 式掃描兩種。前者成像速度較快(約每秒ㄧ張)，而後者可結合「鎖像放大」或「光 子計數」技術大幅提升訊雜比。我們自行開發一套全新的光學成像系統，將鎖相 放大技術與雷射光束掃描式倍頻顯微技術結合，可獲得高訊雜比的動態影像，並 將此系統應用於觀測肌肉細胞之誘發收縮與心肌細胞之自主收縮。本論文首先介 紹實驗系統，並展現雙光子激發螢光訊號的提升，藉以驗證實驗系統之可行性。 接著應用此「鎖相放大掃描式倍頻光學顯微影像技術」觀察肌肉細胞株受電脈衝 刺激誘發之肌纖維收縮，並獲得肌小節收縮頻率與肌小節長度之變化。我們也應 用此系統觀察雞胚胎心肌細胞之自發性收縮，並透過影像分析獲得肌小節長度變 化與肌小節收縮與放鬆速度。我們初步發現肌纖維的位移與肌纖維內部肌小節長 度的改變雖然頻率近似，但在時間上並不完全同步，而有一定的相位差。肌小節 每次收縮的長度變化量也並不固定。在腎上腺素刺激後，肌小節之收縮速度有明 顯增加而放鬆速度則無顯著改變。我們的結果顯示，鎖相放大倍掃描式頻顯微成 像技術極適合應用於研究肌肉細胞的動態過程，除了可應用於研究肌纖維收縮之 基礎生理研究之外，也可應用於探討藥物或毒物對心肌收縮之影響。

Optical microscopy, especially laser scanning microscopy, has been a powerful tool for biomedical research. Concerning laser scanning optical microscopy, a two-dimensional image is constructed by scanning the focus of a laser beam across a fixed sample or vice versa. The frame rate of the former is larger, but the latter allows incorporation of techniques such as lock-in amplification or photon counting to improve signal-to-noise ratio. We have developed a novel lock-in amplified laser scanning second-harmonic generation (SHG) microscopy system, by firstly integrating a pulsed laser into a commercial confocal microscope, and secondly incorporating modulation-demodulation technique into the system. The signal-to-noise ratio is significantly improved while maintaining fast frame rate. The thesis first details the setup of the system including the setting of experiment parameters, and follows with demonstration of the system to image the contraction of two distinct kinds of myocytes: electrically stimulated skeletal muscle cell line and spontaneously contracting chick embryo cardiomyocytes. We show that we can follow the contracting-resting cycle of either electrically induced contraction of skeletal muscles or spontaneous contraction of primary cardiomyocytes. Through image analysis, we can determine the change of sarcomere length during electrically stimulated or spontaneous contraction and examine the effect of hormone on sarcomere contractions.