Tracking of cell surface receptors by fluorescence digital imaging microscopy using a charge-coupled device camera....

A fluorescence imaging system, based on using a cooled slow-scan CCD camera, has been developed for tracking receptors on the surfaces of living cells. The technique is applicable to receptors for particles such as lipoproteins and viruses that can be labeled with a few tens of fluorophores. The positions of single particles in each image are determined to within 25 nm by fitting the fluorescence distribution to a two-dimensional Gaussian function. This procedure also provides an accurate measure of intensity, which is used as a tag for automated tracking of particles from frame to frame. The method is applied to an investigation of the mobility of receptors for LDL and influenza virus particles on human dermal fibroblasts at 4 degrees C. In contrast to previous studies by FRAP (fluorescence recovery after photo-bleaching), it is found that receptors have a low but measurable mobility at 4 degrees C. Analysis of individual particle tracks indicates that whilst some receptors undergo random diffusion, others undergo directed motion (flow) or diffusion restricted to a domain. A procedure is proposed for subdividing receptors according to their different types of motion and hence determining their motional parameters. The finding that receptors are not completely immobilised at 4 degrees C is significant for studies of receptor distributions performed at this temperature.