Morrison Ian E G, Karakikes Ioannis, Barber Rosamund E, Fernández Nelson, Cherry Richard J
Department of Biological Sciences, University of Essex, Colchester, CO4 3SQ, UK.
Biophys J. 2003 Dec;85(6):4110-21. doi: 10.1016/S0006-3495(03)74823-1.
Single particle fluorescence imaging (SPFI) uses the high sensitivity of fluorescence to visualize individual molecules that have been selectively labeled with small fluorescent particles. The positions of particles are determined by fitting the intensity profile of their images to a 2-D Gaussian function. We have exploited the positional information obtained from SPFI to develop a method for detecting colocalization of cell surface molecules. This involves labeling two different molecules with different colored fluorophores and determining their positions separately by dual wavelength imaging. The images are analyzed to quantify the overlap of the particle images and hence determine the extent of colocalization of the labeled molecules. Simulated images and experiments with a model system are used to investigate the extent to which colocalization occurs from chance proximity of randomly distributed molecules. A method of correcting for positional shifts that result from chromatic aberration is presented. The technique provides quantification of the extent of colocalization and can detect whether colocalized molecules occur singly or in clusters. We have obtained preliminary data for colocalization of molecules on intact cells. Cells often exhibit particulate autofluorescence that can interfere with the measurements; a method for overcoming this problem by triple wavelength imaging is described.
单粒子荧光成像（SPFI）利用荧光的高灵敏度来可视化已被小荧光颗粒选择性标记的单个分子。通过将粒子图像的强度分布拟合到二维高斯函数来确定粒子的位置。我们利用从SPFI获得的位置信息开发了一种检测细胞表面分子共定位的方法。这包括用不同颜色的荧光团标记两种不同的分子，并通过双波长成像分别确定它们的位置。对图像进行分析以量化粒子图像的重叠，从而确定标记分子的共定位程度。使用模拟图像和模型系统实验来研究随机分布分子的偶然接近导致共定位的程度。提出了一种校正色差引起的位置偏移的方法。该技术可对共定位程度进行量化，并能检测共定位分子是单独出现还是成簇出现。我们已经获得了完整细胞上分子共定位的初步数据。细胞常常表现出颗粒状自发荧光，这可能会干扰测量；描述了一种通过三波长成像克服这个问题的方法。
