3D time resolved multiphoton fluorescence lifetime imaging microscopy of nano-crystalline agricultural treatments on living plant tissue
3D time resolved multiphoton fluorescence lifetime imaging microscopy of nano-crystalline agricultural treatments on living plant tissue
复制标题
活体植物组织纳米晶农业处理的 3D 时间分辨多光子荧光寿命成像显微镜
DOI：
10.1117/12.2650736
复制
发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Howard, Scott
中科院分区：
文献类型：
--
作者：
Yuan, Xiaotong;Mannam, Varun;Howard, Scott
分子成像工具可以对植物组织微环境中的植物代谢和外部农业处理的影响进行成像，对于进一步了解植物生物学和优化新农产品配方具有重要意义。质谱是植物生物学家常用的工具，无法解析叶子表面的纳米晶体活性成分 (AI)，也无法实现活体植物的 3D 分子成像。为了解决这个问题，将多光子显微镜 (MPM) 和荧光寿命成像显微镜 (FLIM) 相结合，以在除草剂处理后实现 AI 和内在蛋白质/色素（例如叶绿素和/或胞质 NADH）的亚细胞深度分辨荧光寿命。在这里，我们提出了一种使用定制设计的高速 MPM-FLIM 系统“Instant FLIM”的方法，通过应用外部处理，对光学厚且高度散射的植物样品中的内在蛋白质和色素实现实时、未标记的 3D 功能分子成像。为了验证 MPM-FLIM 测量植物组织内内在蛋白质和色素的能力，我们展示了未标记的早熟禾叶片样品的结果。为了演示 3D 分子植物组织和农业 AI 纳米晶体沉积和形成的同步成像，我们通过将商业除草剂产品应用于伽马草叶片样品来评估 MPM-FLIM 的性能。此外，为了测量活体植物组织内除草剂诱导的细胞水平功能反应，对含有除草剂的大麻罗布麻叶进行了 3D 时间分辨分子 MPM-FLIM 成像。结果表明，MPM-FLIM 能够对无标记活体植物组织进行 3D 同步功能成像，并对植物组织内 AI 纳米晶体的位置和形成进行定量测量。
Molecular imaging tools that can image plant metabolism and effects of external agricultural treatments in the micro-environment of plant tissues are significant for further understanding plant biology and optimizing the formulation of new agricultural products. Mass spectrometry, a common tool used by plant biologists, is unable to resolve nano-crystalline active ingredients (AIs) on the leaf surface nor achieve 3D molecular imaging of living plants. To address that, multiphoton microscopy (MPM) and fluorescence lifetime imaging microscopy (FLIM) are combined to achieve sub-cellular, depth-resolved fluorescence lifetime of both AIs and intrinsic proteins/pigments (e.g., chlorophyll and/or cytosolic NADH) after the herbicide treatment application. Here we present a method using a custom-designed, high-speed MPM-FLIM system, “Instant FLIM”, to achieve real-time, unlabeled 3D functional molecular imaging of intrinsic proteins and pigments in optically thick and highly scattering plant samples with the application of external treatments. To validate the capability of MPM-FLIM to measure intrinsic proteins and pigments within plant tissues, we present the results of unlabeled bluegrass blades samples. To demonstrate simultaneous imaging of 3D molecular plant tissue and the agricultural AI nano-crystals deposition and formation, we evaluate the performance of the MPM-FLIM by applying commercial herbicide product to gamagrass blade sample. Additionally, to measure the herbicide-induced cellular-level functional responses within living plant tissues, 3D time-resolved molecular MPM-FLIM imaging of hemp dogbane leaf with herbicide is performed. Results demonstrate MPM-FLIM is capable of 3D simultaneous functional imaging of label-free living plant tissues and the quantitative measurements of the location and formation of AI nanocrystals within the plant tissues.
影响因子：
3.5
作者：
Datta, Rupsa;Heaster, Tiffany M.;Skala, Melissa C.
通讯作者：
Skala, Melissa C.
克服频域荧光寿命成像显微镜空间分辨率的基本限制
DOI：
--
发表时间：
2021
期刊：
BiOS
影响因子：
--
作者：
Xiaotong Yuan;Varun Mannam;Yide Zhang;S. Howard
通讯作者：
S. Howard