Hoppe Adam D, Shorte Spencer L, Swanson Joel A, Heintzmann Rainer
Department of Microbiology and Immunology, University of Michigan, Ann Arbor, Michigan, USA.
Biophys J. 2008 Jul;95(1):400-18. doi: 10.1529/biophysj.107.125385. Epub 2008 Mar 13.
Analysis of cellular pathways requires concentration measurements of dynamically interacting molecules within the three-dimensional (3D) space of single living cells. Förster resonance energy transfer (FRET) microscopy from widefield, from confocal, and potentially from superresolution microscopes can access this information; however, these measurements are distorted by the inherent 3D blurring of optical imaging, spectral overlap of fluorophores, and detection noise. We propose a mathematical model of these processes and demonstrate, through simulation, how these distortions limit the dynamic range and sensitivity of conventional FRET microscopy. Using this model, we devise and validate a new approach (called 3D-FRET stoichiometry reconstruction, 3DFSR) for reconstructing 3D distributions of bound and free fluorescent molecules. Previous attempts to reconstruct 3D-FRET data relied on sequential spectral unmixing and deconvolution, a process that corrupts the detection statistics. We demonstrate that 3DFSR is superior to these approaches since it simultaneously models spectral mixing, optical blurring, and detection noise. To achieve the full potential of this technique, we developed an instrument capable of acquiring 3D-FRET data rapidly and sensitively from single living cells. Compared with conventional FRET microscopy, our 3D-FRET reconstruction technique and new instrumentation provides orders of magnitude gains in both sensitivity and accuracy wherein sustained high-resolution four-dimensional (x,y,z,t) imaging of molecular interactions inside living cells was achieved. These results verify previous observations that Cdc42 signaling is localized to the advancing margins of forming phagosomes in macrophages.
细胞通路分析需要对单个活细胞三维（3D）空间内动态相互作用的分子进行浓度测量。宽场、共聚焦以及潜在的超分辨率显微镜的Förster共振能量转移（FRET）显微镜可以获取此信息；然而，这些测量会受到光学成像固有的3D模糊、荧光团的光谱重叠以及检测噪声的影响而失真。我们提出了这些过程的数学模型，并通过模拟证明了这些失真如何限制传统FRET显微镜的动态范围和灵敏度。使用该模型，我们设计并验证了一种用于重建结合和游离荧光分子的3D分布的新方法（称为3D-FRET化学计量学重建，3DFSR）。以前重建3D-FRET数据的尝试依赖于顺序光谱解混和去卷积，这一过程会破坏检测统计数据。我们证明3DFSR优于这些方法，因为它同时对光谱混合、光学模糊和检测噪声进行建模。为了充分发挥这项技术的潜力，我们开发了一种能够从单个活细胞中快速、灵敏地获取3D-FRET数据的仪器。与传统FRET显微镜相比，我们的3D-FRET重建技术和新仪器在灵敏度和准确性方面都有数量级的提升，从而实现了对活细胞内分子相互作用的持续高分辨率四维（x,y,z,t）成像。这些结果证实了之前的观察结果，即Cdc42信号定位于巨噬细胞中正在形成的吞噬体的前进边缘。
