In this study, we integrate a single-photon detector array into a confocal laser scanning microscope, enabling the combination of fluorescence-lifetime single-molecule localization microscopy with image scanning microscopy. This unique combination delivers a twofold improvement in lateral localization accuracy for single-molecule localization microscopy (SMLM) and maintains its simplicity. Moreover, the addition of lifetime information from our confocal laser scanning microscope eliminates chromatic aberration, particularly crucial for achieving few-nanometre resolution in SMLM. Our approach, named fluorescence-lifetime image scanning microscopy SMLM, is demonstrated through direct stochastic optical reconstruction microscopy and DNA point accumulation for imaging in nanoscale topography experiments on fluorescently labelled cells, showcasing both resolution enhancement and fluorescence-lifetime multiplexing capabilities.

在这项研究中，我们将单光子探测器阵列集成到共焦激光扫描显微镜中，从而实现了荧光寿命单分子定位显微镜与图像扫描显微镜的结合。这种独特的组合使单分子定位显微镜 (SMLM) 的横向定位精度提高了两倍，并保持了其简单性。此外，我们的共焦激光扫描显微镜添加的寿命信息消除了色差，这对于在 SMLM 中实现几纳米分辨率尤其重要。我们的方法被称为荧光寿命图像扫描显微镜 SMLM，通过直接随机光学重建显微镜和 DNA 点积累在荧光标记细胞的纳米级形貌实验中成像来证明，展示了分辨率增强和荧光寿命多重功能。