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）成像早已被确立为检测蛋白质相互作用和感知生命系统中生理状态的最佳方法。然而，整个生物体的3D体内FRET成像仍然具有高度挑战性，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。本文提出了一种深部组织荧光寿命成像（FLIM）方法，即寿命激发发射矩阵扫描激光光学层析成像（LEEM-SLOT）。在深部组织中执行各向同性亚细胞3D成像，2.在一次扫描中进行多色FRET成像，3.充分分析多色FRET。LEEM-SLOT将允许快速FLIM断层扫描并在4x 4激发发射矩阵中重建寿命图像，其中1-？m各向同性分辨率超过500-？m深度。光谱未混合的LEEM图像的寿命分析将允许使用CFP/GFP/YFP/mCherry和等同物的多个FRET通道的并行可视化。我们将LEEM-SLOT方法应用于两个试点研究：1。在玻璃海鞘心脏祖细胞中的MAP激酶途径激活，和2.斑马鱼肾脏囊肿形成过程中细胞内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，并在活生物体中进行多模式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.