Jang Soohyen, Narayanasamy Kaarjel K, Rahm Johanna V, Saguy Alon, Kompa Julian, Dietz Marina S, Johnsson Kai, Shechtman Yoav, Heilemann Mike
Institute of Physical and Theoretical Chemistry, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.
Institute of Physical and Theoretical Chemistry, IMPRS on Cellular Biophysics, Johann Wolfgang Goethe-University, Frankfurt am Main, Germany.
Biophys Rep (N Y). 2023 Aug 18;3(3):100123. doi: 10.1016/j.bpr.2023.100123. eCollection 2023 Sep 13.
Single-molecule localization microscopy achieves nanometer spatial resolution by localizing single fluorophores separated in space and time. A major challenge of single-molecule localization microscopy is the long acquisition time, leading to low throughput, as well as to a poor temporal resolution that limits its use to visualize the dynamics of cellular structures in live cells. Another challenge is photobleaching, which reduces information density over time and limits throughput and the available observation time in live-cell applications. To address both challenges, we combine two concepts: first, we integrate the neural network DeepSTORM to predict super-resolution images from high-density imaging data, which increases acquisition speed. Second, we employ a direct protein label, HaloTag7, in combination with exchangeable ligands (xHTLs), for fluorescence labeling. This labeling method bypasses photobleaching by providing a constant signal over time and is compatible with live-cell imaging. The combination of both a neural network and a weak-affinity protein label reduced the acquisition time up to ∼25-fold. Furthermore, we demonstrate live-cell imaging with increased temporal resolution, and capture the dynamics of the endoplasmic reticulum over extended time without signal loss.
单分子定位显微镜通过定位在空间和时间上分离的单个荧光团来实现纳米级空间分辨率。单分子定位显微镜的一个主要挑战是采集时间长，这导致通量低，以及时间分辨率差，限制了其在活细胞中可视化细胞结构动态的应用。另一个挑战是光漂白，它会随着时间降低信息密度，并限制活细胞应用中的通量和可用观察时间。为了解决这两个挑战，我们结合了两个概念：首先，我们集成了神经网络DeepSTORM，以从高密度成像数据预测超分辨率图像，这提高了采集速度。其次，我们采用直接蛋白质标签HaloTag7与可交换配体（xHTLs）结合进行荧光标记。这种标记方法通过随时间提供恒定信号来绕过光漂白，并且与活细胞成像兼容。神经网络和弱亲和力蛋白质标签的结合将采集时间减少了约25倍。此外，我们展示了具有更高时间分辨率的活细胞成像，并在延长的时间内捕获内质网的动态而无信号损失。
