Department of Biomedical Engineering, OHSU Center for Spatial Systems Biomedicine, and Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon.
Department of Biomedical Engineering, OHSU Center for Spatial Systems Biomedicine, and Knight Cancer Institute, Oregon Health and Science University, Portland, Oregon.
Biophys J. 2018 Jan 23;114(2):301-310. doi: 10.1016/j.bpj.2017.11.013.
Single-molecule tracking (SMT) offers rich information on the dynamics of underlying biological processes, but multicolor SMT has been challenging due to spectral cross talk and a need for multiple laser excitations. Here, we describe a single-molecule spectral imaging approach for live-cell tracking of multiple fluorescent species at once using a single-laser excitation. Fluorescence signals from all the molecules in the field of view are collected using a single objective and split between positional and spectral channels. Images of the same molecule in the two channels are then combined to determine both the location and the identity of the molecule. The single-objective configuration of our approach allows for flexible sample geometry and the use of a live-cell incubation chamber required for live-cell SMT. Despite a lower photon yield, we achieve excellent spatial (20-40 nm) and spectral (10-15 nm) resolutions comparable to those obtained with dual-objective, spectrally resolved Stochastic Optical Reconstruction Microscopy. Furthermore, motions of the fluorescent molecules did not cause loss of spectral resolution owing to the dual-channel spectral calibration. We demonstrate SMT in three (and potentially more) colors using spectrally proximal fluorophores and single-laser excitation, and show that trajectories of each species can be reliably extracted with minimal cross talk.
单分子追踪 (SMT) 提供了有关潜在生物过程动态的丰富信息，但由于光谱串扰和需要多次激光激发，多色 SMT 一直具有挑战性。在这里，我们描述了一种单分子光谱成像方法，用于使用单激光激发同时对多个荧光物种进行活细胞追踪。使用单个物镜收集视场中所有分子的荧光信号，并将其在位置和光谱通道之间进行分离。然后将两个通道中同一分子的图像组合起来，以确定分子的位置和身份。我们的方法的单物镜配置允许灵活的样品几何形状，并允许使用活细胞 SMT 所需的活细胞孵育室。尽管光子产率较低，但我们实现了与双物镜、光谱分辨的随机光学重建显微镜相当的优异空间 (20-40nm) 和光谱 (10-15nm) 分辨率。此外，由于双通道光谱校准，荧光分子的运动不会导致光谱分辨率的损失。我们使用光谱相近的荧光染料和单激光激发在三种（潜在更多种）颜色中展示了 SMT，并表明可以可靠地提取每种物质的轨迹，并且串扰最小。
