Traeger Jeremiah, Yang Mengran, Stacey Gary, Orr Galya, Hu Dehong
Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, Washington.
Division of Plant Sciences and Technology, University of Missouri, Columbia, Missouri.
Biophys J. 2025 Feb 4;124(3):574-585. doi: 10.1016/j.bpj.2024.12.028. Epub 2024 Dec 30.
Plant leaf tissues are difficult to image via fluorescence microscopy due to the presence of chlorophyll and other pigments, which provide large background fluorescence. Lattice light-sheet microscopy offers the advantage of using Bessel beams to illuminate a thin focal region of interest for microscopy, allowing for the excitation of fluorescent molecules within this region without surrounding chlorophyll-like objects outside of the region of interest. Here, we apply STORM super-resolution techniques to observe receptor-like kinases in Arabidopsis thaliana leaf cells. By applying this technique with lattice light-sheet microscopy, we can localize immune-response proteins at sub-100-nm length scales and reconstruct three-dimensional locations of proteins within individual leaf cells. Using this technique, we observed the effect of the ATP and flg22 elicitors, where we observed a significant degree of internalization of cognate receptors P2K1 and FLS2. We were also able to similarly observe differences in colocalization due to stimulation with these elicitors, whereby we observe proteins on the membrane becoming less colocalized as a result of stimulation, suggesting an immune-response mechanism involving receptor internalization via distinct pathways. These data show lattice light-sheet microscopy's capabilities for imaging tissue with problematic background fluorescence that otherwise makes super-resolution fluorescence microscopy difficult.
由于叶绿素和其他色素的存在，植物叶片组织难以通过荧光显微镜成像，这些色素会产生大量背景荧光。晶格光片显微镜的优势在于利用贝塞尔光束照亮感兴趣的薄聚焦区域以进行显微镜观察，从而能够激发该区域内的荧光分子，而不会激发感兴趣区域之外的类似叶绿素的物体。在此，我们应用STORM超分辨率技术来观察拟南芥叶片细胞中的类受体激酶。通过将该技术与晶格光片显微镜结合使用，我们可以在亚100纳米的长度尺度上定位免疫反应蛋白，并重建单个叶片细胞内蛋白质的三维位置。使用该技术，我们观察了ATP和flg22诱导子的作用，在此过程中我们观察到同源受体P2K1和FLS2有显著程度的内化。我们还能够类似地观察到由于这些诱导子刺激导致的共定位差异，由此我们观察到膜上的蛋白质因刺激而共定位减少，这表明存在一种涉及通过不同途径进行受体内化的免疫反应机制。这些数据展示了晶格光片显微镜在对具有有问题背景荧光的组织进行成像方面的能力，否则这种背景荧光会使超分辨率荧光显微镜观察变得困难。
