III. Institute of Physics - Biophysics, Georg August University, 37077 Göttingen, Germany.
Laboratory of Supramolecular Chemistry, EPFL SB ISIC LCS, BCH 3307, CH-1015 Lausanne, Switzerland.
Nano Lett. 2022 Aug 10;22(15):6454-6461. doi: 10.1021/acs.nanolett.2c01586. Epub 2022 Jul 6.
A recent addition to the toolbox of super-resolution microscopy methods is fluorescence-lifetime single-molecule localization microscopy (FL-SMLM). The synergy of SMLM and fluorescence-lifetime imaging microscopy (FLIM) combines superior image resolution with lifetime information and can be realized using two complementary experimental approaches: confocal-laser scanning microscopy (CLSM) or wide-field microscopy. Here, we systematically and comprehensively compare these two novel FL-SMLM approaches in different spectral regions. For wide-field FL-SMLM, we use a commercial lifetime camera, and for CLSM-based FL-SMLM we employ a home-built system equipped with a rapid scan unit and a single-photon detector. We characterize the performances of the two systems in localizing single emitters in 3D by combining FL-SMLM with metal-induced energy transfer (MIET) for localization along the third dimension and in the lifetime-based multiplexed bioimaging using DNA-PAINT. Finally, we discuss advantages and disadvantages of wide-field and confocal FL-SMLM and provide practical advice on rational FL-SMLM experiment design.
最近，超分辨率显微镜方法的工具箱中又增加了一种新方法，即荧光寿命单分子定位显微镜（FL-SMLM）。SMLM 和荧光寿命成像显微镜（FLIM）的协同作用将优异的图像分辨率与寿命信息结合在一起，可以通过两种互补的实验方法来实现：共聚焦激光扫描显微镜（CLSM）或宽场显微镜。在这里，我们系统而全面地比较了这两种在不同光谱区域的新型 FL-SMLM 方法。对于宽场 FL-SMLM，我们使用商业寿命相机，而对于基于 CLSM 的 FL-SMLM，我们使用配备快速扫描单元和单光子探测器的自制系统。我们通过将 FL-SMLM 与金属诱导能量转移（MIET）结合，沿第三个维度进行定位，以及在基于寿命的 DNA-PAINT 多重生物成像中，对这两个系统在 3D 中定位单荧光体的性能进行了表征。最后，我们讨论了宽场和共聚焦 FL-SMLM 的优缺点，并提供了关于合理设计 FL-SMLM 实验的实用建议。
