High-resolution multi-color FLIM-FRET tomography of whole organism

描述(由申请人提供)：共振能量转移(FRET)成像长期以来一直被认为是检测蛋白质相互作用和感知生命系统生理状态的最佳方法。然而，整个生物体的3D活体FRET成像仍然具有很高的挑战性，因为现有的显微镜方法不能进行深穿透的亚细胞多通道FRET成像。我们提出了一种深部组织荧光寿命成像(FLIM)方法，称为寿命激发发射矩阵扫描激光光学层析成像(LEEM-SLOT)，用于：1.在深部组织中进行各向同性亚细胞三维成像；2.一次扫描完成多色FRET成像；3.全面分析多色FRET。LEEM槽将允许在4x4激发发射矩阵中以1？M各向同性分辨率在500-？m深度上进行快速薄膜层析成像和寿命图像重建。对光谱未混合的LEEM图像的寿命分析将允许并行可视化具有CFP/GFP/YFP/mCherry和等价物的多个FRET通道。我们将把LEEM-Slot方法应用于两个先导性研究：1.斑马鱼心脏祖细胞MAP-Kinase通路的激活；2.活斑马鱼肾脏成囊过程中细胞内cAMP水平的变化。通过这些研究，我们期望建立LEEM-Slot方法作为一种新的工具，用于研究活胚胎或组织中信号通路的区域激活，并在活生物中进行多模式FRET生物传感。

DESCRIPTION (provided by applicant): Resonant Energy Transfer (FRET) imaging has long been established as the best method for detecting protein interaction and sensing physiological state in living systems. However, 3D in vivo FRET imaging of whole organism remains highly challenging, because existing microscopy methods cannot perform deep- penetrating sub-cellular multi-channel FRET imaging. We propose to develop a deep tissue Fluorescence lifetime imaging (FLIM) method, named Lifetime Excitation-Emission Matrix Scanning Laser Optical Tomography (LEEM-SLOT) to: 1. Perform isotropic sub-cellular 3D imaging in deep tissue, 2. Perform multi- color FRET imaging in one scan, 3. Fully analyze multi-color FRET. LEEM-SLOT will permit fast FLIM tomography and reconstruction of lifetime images in 4x4 excitation emission matrices with 1-?m isotropic resolution over 500-?m depth. Lifetime analysis of spectrally unmixed LEEM images will allow parallel visualization of multiple FRET channels with CFP/GFP/YFP/mCherry and equivalents. We will apply the LEEM-SLOT method to two pilot studies of: 1. Map Kinase pathway activation in Ciona heart progenitor cells, and 2. Intracellular cAMP level during renal cystogenesis in live zebrafish. Through these studies we expect to establish the LEEM-SLOT method as a novel tool for studying the regional activation of signaling pathways in live embryos or tissues, and performing multi-modal FRET biosensing in live organisms.