Fig. 5.

FCS recordings from the plasma membrane of choroid plexus epithelial cells labeled with 2C-Fab-GFP, and the plasma membrane of transfected HEK293 cells expressing monomeric CD-86 with one GFP tag (CD-86–GFP) or two GFP tags (CD-86–GFP-GFP). (A) Fluorescence intensity traces for one 10-second observation period. (B) Autocorrelation analysis of the fluorescence intensity traces. The red line represents the autocorrelation of the observed fluorescence signal, and the green line represents the fit to a two-component model: a fast component (on the order of 300 microseconds) related to the photo-physical properties of GFP, and a slower component (on the order of 40 milliseconds) representing the translational diffusion of fluorescence-tagged receptors in the plasma membrane. Dividing the average photon count rate (kilohertz) determined from the fluorescence intensity trace (A) by the number of fluorescent molecules determined from the amplitude of the autocorrelation curve (B) predicts the average molecular brightness of the sample. (C) Photon counting histograms of the corresponding FCS recordings. To generate the histograms, each 10-second fluorescence intensity trace (A) was broken down into one million 10-microsecond intervals or bins (PCH bin time = 10 microseconds). The number of bins is plotted on the y-axis and photon counts on the x-axis. (D) Residuals of the PCH curve fit to a one-component model. The residuals of the curve fit are less than 2 standard deviations and are randomly distributed about zero, indicating a good fit to the selected model.