We develop a multidimensional fluorescence imaging technique by implementing a wide‐field time‐gated fluorescence lifetime imaging into digital scanned laser light‐sheet microscopy (FLIM‐DSLM) to measure 3D fluorescence lifetime distribution in mesoscopic specimens with high resolution. This is achieved by acquiring a series of time‐gated images at different relative time delays with respect of excitation pulses at different depths. The lifetime is determined for each voxel by iteratively fitting to single exponential decay. The performance of the developed system is evaluated with the measurements of a lifetime reference Rhodamine 6G solution and a subresolution fluorescent bead phantom. We also demonstrate the application performances of this system to ex vivo and in vivo imaging of Tg(kdrl:EGFP) transgenic zebrafish embryos, illustrating the lifetime differences between the GFP signal and the autofluorescence signal. The results show that FLIM‐DSLM can be used for sample size up to a few millimetres and can be utilised as a powerful and robust method for biomedical research, for example as a readout of protein–protein interactions via Förster resonance energy transfer.

中文翻译：

---

具有宽视场时间门控成像的数字扫描激光光片荧光寿命显微镜

我们通过在数字扫描激光光片显微镜 (FLIM-DSLM) 中实施宽视场时间门控荧光寿命成像来开发多维荧光成像技术，以高分辨率测量细观样品中的 3D 荧光寿命分布。这是通过相对于不同深度的激发脉冲以不同的相对时间延迟获取一系列时间门控图像来实现的。通过迭代拟合单个指数衰减来确定每个体素的寿命。所开发系统的性能通过对寿命参考罗丹明 6G 溶液和亚分辨率荧光珠体模的测量进行评估。我们还展示了该系统在 Tg(kdrl:EGFP) 转基因斑马鱼胚胎的离体和体内成像中的应用性能，说明 GFP 信号和自发荧光信号之间的寿命差异。结果表明，FLIM-DSLM 可用于高达几毫米的样本大小，并可用作生物医学研究的强大而可靠的方法，例如通过 Förster 共振能量转移读取蛋白质-蛋白质相互作用。