METHOD OF FORMING ENHANCED SUPER-RESOLUTION IMAGE

專利類型

發明

專利國別 (專利申請國家)

美國

專利申請案號

16/547,607

專利證號

US 10,994,993

專利獲證名稱

METHOD OF FORMING ENHANCED SUPER-RESOLUTION IMAGE

專利所屬機關 (申請機關)

國立中央大學

獲證日期

2021/05/04

技術說明

為了減少在使用單分子定位顯微術時對細胞樣本的光損害影響，此發明中研發表面電漿偏極子增強螢光效應，可同時降低激發功率密度並維持單分子定位顯微影像的定位解析度。基於單分子定位顯微術的超解析度影像，同時結合表面電漿偏極子增強閃爍螢光團之微弱螢光訊號，可實現觀測細胞聚集吸附相關蛋白的奈米解析度之定位資訊。具有奈米金屬層沉積的玻璃基板可被用來誘導金屬層表面上表面電漿偏極子增強的場以及耦合放射效應，並顯著地增強螢光團的螢光信號。使用物鏡架構之全反射螢光激發形式時，相較於非增強之基板，於表面電漿偏極子增強之基板的螢光團，其螢光信號強300％以上並具有更多閃爍螢光分子表現。具有25±11nm的空間解析度的定位成像只需31.6W cm -2的照射功率密度，相較於傳統單分子定位顯微術所需幾kW cm2.等級，大幅度的降低生物樣本之照射功率密度。對於單分子定位顯微術，這種低照度功率密度將有效的降低生物樣本的光毒性。再者，此發明中所提出的策略，在金屬表面上可提供均勻分佈的表面電漿偏極子增強場，使得在觀測聚螢光標記之蛋白質的空間分佈資訊時，將不會受到由不均勻分佈的增強場效應所引起之干擾，進而產生的誤差。因此，表面電漿偏極子增強的單分子定位顯微術將是研究活細胞中近膜蛋白質之動力學表現和組成分佈的有效方法。 To reduce photodamage to cell specimens when using single-molecule localization microscopy, surface plasmon polariton (SPP)-enhanced fluorescence was demonstrated to lower the excitation power density and maintain the positioning resolution of the single-molecule localization. Super-resolution imaging based on single-molecule localization microscopy combined with the SPP-enhanced fluorescence of blinking fluorophores was demonstrated to visualize the nanoscale-level positioning information of cell-adhesion-associated proteins. Glass substrates with a deposited metal layer were utilized to induce a SPP-enhanced field on the metal surface and significantly strengthen the fluorescence signals of the fluorophores. Using objective-type TIRF excitation, the fluorescence signals of the fluorophores were more than 300% stronger for the SPP-enhanced substrate compared with those of the non-enhanced substrate, and more blinking events occurred. The illumination power density for localization imaging at a spatial resolution of 25 ± 11 nm was 31.6 W cm-2. This low illumination power density will facilitate the reduction of phototoxicity of the biospecimens for single-molecule localization imaging. The proposed strategy provides a uniform distribution of the SPP-enhanced field on the metal surface, enabling visualization of the spatial distribution of labeled proteins without interference caused by the enhanced field distribution. In conclusion, SPP-enhanced single-molecule localization microscopy is a useful approach for investigating the dynamics and organization of proteins close to the membrane in living cells.

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