結構照明螢光顯微術與生物應用

Structured Illumination Fluorescence Microscopy and Its Biological Applications

張博睿(Bo-Jui Chang) ； 周俐君(Li-Jun Chou) ； 江素玉(Su-Yu Chiang)

《科儀新知》 180期 (2011/02) Pp. 28-38

https://doi.org/10.29662/IT.201102.0003

摘要

螢光顯微術具有高靈敏度、高分子鑑別度、同時偵測多重色和活體細胞影像等優點，但空間解析度受光繞射特性的限制，無法解析距離比1/2螢光波長還要靠近的兩物體，產生模糊且重疊的影像。近期以來許多先進螢光顯微術利用非線性光學、光干涉或特殊螢光染劑的光激發特性，已成功地突破繞射極限，提高螢光影像的二維或三維解析度，解析出更細微的細胞結構，分辨出多種蛋白質分子的分布和交互作用等性質。國家同步輻射中心於2010年建立一套高解析結構照明螢光顯微實驗系統，主要利用不同角度的三維結構照明圖案激發螢光樣品，再由螢光影像解析出高頻率的新資訊，提供近兩倍的三維解析度。本文將簡介結構照明螢光顯微術高解析影像的重建原理、可快速調變照明圖案相位和方位的顯微實驗系統，以及在生物研究的應用。

並列摘要

Fluorescence microscopy enables biologists to observe the fine structures of specimens with great contrast and specificity through fluorescent labeling; however, the spatial resolution is limited by the diffraction nature of light. Several super-resolution fluorescence microscopic techniques related to confocal or wide-field microscopy have been developed based on various excitation strategies. Among them, structured illumination fluorescence microscopy (SIFM) that illuminates specimens with structured light, rather than the uniform light used in a wide-field microscope, can attain a doubled resolution in three dimensions. At NSRRC, we have developed a 3D-SIFM system that uses a spatial light modulator (SLM) to shift and to rotate the illumination pattern rapidly and precisely, without mechanical calibration. Simulations of the optical transfer function of this SIFM system are also performed for Wiener deconvolution to improve image resolution. Here, we describe the concept of SIFM, introduce our newly launched SLM-based 3D-SIFM system, and demonstrate the prospective application to biological systems.