Figure 2.
Figure 2. Counting the numbers of apparent monomer/dimer fluorescent spots of AlexaFP in a single-frame TIRF image. (A) A representative single-frame TIRF image of single AlexaFP molecules bound to D71A, observed at 7 min after the addition of 6 nM AlexaFP. Individual fluorescent spots were identified by taking the cross-correlation of the observed image with a reference image of a single FM spot. Yellow arrowheads and red arrows indicate the spots with monomeric and dimeric intensities (< and >18 AU in D), respectively. (B) This image cross-correlation method, in addition to identifying the fluorescent spots, finds the local peaks in the correlation image, determining whether an observed spot actually represents one unresolvable spot or two resolvable spots (bottom; spatial resolution). Here, a typical 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 x axis. Top, raw images; bottom, cross-correlation images, with red dots showing local peaks; typical examples using D71A-mGFP), and for each shift distance, the result of one or two peaks was registered in the cross-correlation images (bottom). (C) Determining the spatial resolution for single FM imaging. The process of B was repeated for 300 single FM spots with monomeric signal intensities (using images as in A). Then, for each shift distance, we obtained the percentage of fluorescent spots judged to be two space-resolved spots (standard error given as cell-to-cell variations, ncell = 6), which was plotted as a function of the shift distance. The threshold shift distance was ∼220 (219 ± 9.0) nm for mGFP. For the intensity-dependence of spatial resolution, see Materials and methods (“Determining the spatial resolution…”). (D) The distributions of the signal intensities of individual AlexaFP-D71A spots, fitted with the sum of the two Gaussian functions: one is consistent with single AlexaFP molecules with a peak value of ∼12 AU (those bound to the coverslip, bottom); the other is a smaller, broader peak ∼24 AU (9.7 and 23% of the spots in the medium containing 1 and 6 nM AlexaFP, respectively; top and middle).

Counting the numbers of apparent monomer/dimer fluorescent spots of AlexaFP in a single-frame TIRF image. (A) A representative single-frame TIRF image of single AlexaFP molecules bound to D71A, observed at 7 min after the addition of 6 nM AlexaFP. Individual fluorescent spots were identified by taking the cross-correlation of the observed image with a reference image of a single FM spot. Yellow arrowheads and red arrows indicate the spots with monomeric and dimeric intensities (< and >18 AU in D), respectively. (B) This image cross-correlation method, in addition to identifying the fluorescent spots, finds the local peaks in the correlation image, determining whether an observed spot actually represents one unresolvable spot or two resolvable spots (bottom; spatial resolution). Here, a typical 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 x axis. Top, raw images; bottom, cross-correlation images, with red dots showing local peaks; typical examples using D71A-mGFP), and for each shift distance, the result of one or two peaks was registered in the cross-correlation images (bottom). (C) Determining the spatial resolution for single FM imaging. The process of B was repeated for 300 single FM spots with monomeric signal intensities (using images as in A). Then, for each shift distance, we obtained the percentage of fluorescent spots judged to be two space-resolved spots (standard error given as cell-to-cell variations, ncell = 6), which was plotted as a function of the shift distance. The threshold shift distance was ∼220 (219 ± 9.0) nm for mGFP. For the intensity-dependence of spatial resolution, see Materials and methods (“Determining the spatial resolution…”). (D) The distributions of the signal intensities of individual AlexaFP-D71A spots, fitted with the sum of the two Gaussian functions: one is consistent with single AlexaFP molecules with a peak value of ∼12 AU (those bound to the coverslip, bottom); the other is a smaller, broader peak ∼24 AU (9.7 and 23% of the spots in the medium containing 1 and 6 nM AlexaFP, respectively; top and middle).

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