Institute of Physical and Theoretical Chemistry, Goethe-University Frankfurt, Max-von-Laue Str. 7, 60438, Frankfurt, Germany.
Third Institute of Physics-Biophysics, Georg August University, 37077, Göttingen, Germany.
Angew Chem Int Ed Engl. 2021 Mar 15;60(12):6310-6313. doi: 10.1002/anie.202013166. Epub 2021 Feb 3.
Super-resolution optical fluctuation imaging (SOFI) is a super-resolution microscopy technique that overcomes the diffraction limit by analyzing intensity fluctuations of statistically independent emitters in a time series of images. The final images are background-free and show confocality and enhanced spatial resolution (super-resolution). Fluorophore photobleaching, however, is a key limitation for recording long time series of images that will allow for the calculation of higher order SOFI results with correspondingly increased resolution. Here, we demonstrate that photobleaching can be circumvented by using fluorophore labels that reversibly and transiently bind to a target, and which are being replenished from a buffer which serves as a reservoir. Using fluorophore-labeled short DNA oligonucleotides, we labeled cellular structures with target-specific antibodies that contain complementary DNA sequences and record the fluctuation events caused by transient emitter binding. We show that this concept bypasses extensive photobleaching and facilitates two-color imaging of cellular structures with SOFI.
超分辨率光学波动成像（SOFI）是一种超分辨率显微镜技术，通过分析时间序列图像中统计独立发射器的强度波动来克服衍射极限。最终的图像是无背景的，并显示共聚焦和增强的空间分辨率（超分辨率）。然而，荧光漂白是记录允许更高阶 SOFI 结果计算的长时间序列图像的关键限制，相应地提高了分辨率。在这里，我们证明可以通过使用可逆和瞬时结合到靶标的荧光标记物来避免荧光漂白，并且从作为储备库的缓冲液中补充这些标记物。使用荧光标记的短 DNA 寡核苷酸，我们用含有互补 DNA 序列的针对特定靶标的抗体标记细胞结构，并记录由瞬态发射器结合引起的波动事件。我们表明，该概念绕过了广泛的荧光漂白，并促进了具有 SOFI 的细胞结构的双色成像。
