Biology Reference
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20.3 DATA ANALYSIS
20.3.1 Determination of receptor monomer, dimer, and oligomer
fractions in single-color experiments
This section describes a method for measuring fractions of receptor monomers, di-
mers, and oligomers by determining fluorescence intensities of individual spots of
receptors. A typical single-frame image of single molecules of fluorescently labeled
FPR is shown in Fig. 20.4 A. Fluorescent spots in the image are identified using a
FIGURE 20.4
Identification of individual spots. (A) Typical single-frame total internal reflection image of
single FPR molecules labeled with Alexa594-formyl peptide. Individual spots are identified by
taking the cross correlation of the observed image with a reference image of a single-
fluorescent-molecule spot. Arrowheads and arrows indicate the spots with monomeric and
dimeric intensities, respectively. (B) The image cross correlation method, in addition to
identifying the fluorescent spots, finds the local peaks in the correlation image, determining
whether an observed spot represents one unresolvable spot or two resolvable spots
(bottom, spatial resolution). A representative image of a single-molecule intensity spot is
superimposed on itself but at systematically varied shift distances between the two images
(from 0 to 300 nm, every 10 nm along the
-axis). Top panel shows raw images; the bottom
panel displays cross correlation images, with dots indicating local peaks. For each shift
distance, the result of one or two peaks is registered in the cross correlation images (bottom).
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