共時無色相差雙色單分子定位顯微技術之設計與實驗

Design and Experiment for the Chromatic Aberration Free Two-Color Single-Molecule Localization Microscopy

王嘉會(Jia-Hui Wang)

指導教授 ： 楊東霖

國立臺灣大學/電機資訊學院/生醫電子與資訊學研究所/碩士(2021年)

https://doi.org/10.6342/NTU202101837

摘要

單分子定位顯微鏡 (SMLM) 技術的最新發展使研究人員能夠在納米尺度上研究大分子結構。超分辨率技術的分辨率通過有效地銳化點擴散函數打破了光學顯微鏡的衍射極限。 單分子定位顯微鏡在眾多超解析方法中實現了十至二十納米的卓越分辨率，並從包含數十萬個定位的點雲數據集重建了不受到衍射限制的超解析影像。能夠對蛋白質進行共定位分析的多色光學顯微分析對研究者來說至關重要。儘管已經有許多用於多色成像的方法被提出，但其中大多數都需要多個激發波長和收光波段，通過連續採集或光譜分解。
目前尚未有技術可以在單一激發波長和收光波段，同時獲得多色的無色像差超解析影像。本研究提出了一種基於dSTORM（direct stochastic optical reconstruction microscopy）上的共時雙色成像策略，具有單個收光波段，可最效地減少色像差。本研究闡述了 Alexa fluor 647 和 ATTO647N 的光動力學，並開發了一種數據驅動的螢光染料分辨方法。此外，這種方法可以被應用於任何 dSTORM 成像系統，而無需額外的光學組件來分離重疊光譜內的螢光團。

關鍵字

dSTORM ； 超分辨率 ； 共時 ； 無像差 ； 多色成像

並列摘要

Recent developments in single-molecule localization microscopy (SMLM) techniques allow researchers to study macromolecular structures at the nanometer scale. The resolution of superresolution techniques breaks the diffraction limit of light microscopy by effectively sharpening the point spread function. SMLM achieves a superior resolution of 10-20nm among many superresolution methods and reconstructs a diffraction-unlimited image from a point cloud data set that contains hundred thousands of localizations. The multicolor light microscopy capable of performing co-localization analysis of proteins is of crucial importance. Although many approaches for multicolor imaging had been proposed, most of them required multiple light sources and detection windows through sequential acquisition, or spectral unmixing. A simple, simultaneous multicolor imaging free of chromatic aberration has not yet been possible.Here, this study proposes a simultaneous two-color imaging strategy for dSTORM (direct stochastic optical reconstruction microscopy) with a single detection window which minimizes chromatic aberration. The study characterized the photo-switching kinetics of Alexa Fluor 647(AF647) and ATTO647N and developed a data-driven approach for dye separation. Moreover, this method can be applied to any dSTORM imaging system without the need for extra optical components to separate fluorophores within an overlapped spectrum.

並列關鍵字

dSTORM ； superresolution ； simultaneous ； aberration free ； multicolor imaging

參考文獻

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