Department of Molecular Medicine for Pathogenesis, Graduate School of Medicine, Ehime University, Matsuyama, Japan.
Translational Research Center, Ehime University Hospital, Toon, Japan.
Nat Commun. 2021 May 20;12(1):2979. doi: 10.1038/s41467-021-23249-y.
Two-photon excitation can lower phototoxicity and improve penetration depth, but its narrow excitation range restricts its applications in light-sheet microscopy. Here, we propose simple illumination optics, a lens-axicon triplet composed of an axicon and two convex lenses, to generate longer extent Bessel beams. This unit can stretch the beam full width at half maximum of 600-1000 μm with less than a 4-μm waist when using a 10× illumination lens. A two-photon excitation digital scanned light-sheet microscope possessing this range of field of view and ~2-3-μm axial resolution is constructed and used to analyze the cellular dynamics over the whole body of medaka fish. We demonstrate long-term time-lapse observations over several days and high-speed recording with ~3 mm volume per 4 s of the embryos. Our system is minimal and suppresses laser power loss, which can broaden applications of two-photon excitation in light-sheet microscopy.
双光子激发可以降低光毒性并提高穿透深度，但它的窄激发范围限制了其在光片显微镜中的应用。在这里，我们提出了一种简单的照明光学系统，由轴棱锥和两个凸透镜组成的透镜-轴棱锥三重结构，可以产生更长的贝塞尔光束。当使用 10×照明透镜时，该单元可以将光束的半最大值全宽拉伸到 600-1000μm，而腰部直径小于 4μm。构建了具有这种视场范围和 ~2-3-μm 轴向分辨率的双光子激发数字扫描光片显微镜，并用于分析斑马鱼整个身体的细胞动力学。我们演示了几天的长时间延时观察和以 ~3mm 体积/4s 的速度对胚胎进行高速记录。我们的系统是最小的，可以抑制激光功率损耗，从而拓宽双光子激发在光片显微镜中的应用。
