全反射螢光顯微術(TIRFM)的奈米等級解析度和高訊號／背景值的特性，可觀察溶液態中的單分子螢光影像和光譜。本文主旨在說明生物單分子分析在顯微鏡之外的其他設備和處理可以很簡單。生物樣品和玻璃表面經過簡單處理，配合流體幫浦，這種簡易組合就可以利用全反射螢光顯微鏡研究生物單分子；最後，以蛋白質－核酸的交互作用做為例子，說明這種簡易組合在生物單分子動力學的應用。

Total internal reflection fluorescence microscopy (TIRFM) can produce an image and spectrum at a nanometer resolution and with a high signal/noise ratio. This technique is applied to single biomolecule analysis in a growing trend. This text will introduce the basic principle of TIRFM and the techniques combined to TIRFM. In addition, the interaction of protein-nucleic acid will demonstrate the application of TIRFM to single-molecule assay in biological research.