Precise Positioning of Myosin VI on Endocytic Vesicles In Vivo
Figure 5
Steady-State Fluorescence Emission and Anisotropy of Cells Expressing GFP-CBD and CBD-GFP
(A) Steady-state GFP fluorescence emission intensity images (left) and anisotropy images (right) for a cell expressing GFP-CBD. Rectangles in the upper images indicate the region displayed in the lower images. To reduce noise, two–nearest-neighbor averaging was applied to the parallel- and perpendicular-polarization emission intensity images before calculating the total emission intensity and anisotropy images (see Materials and Methods). The units of the total intensity image are in photons/pixel, where photon count is integrated over the pixel residence time of the measurement (102 μs for all measurements). A color map was applied to the anisotropy image and is shown to the right of the images.
To improve contrast of the fluorescence emission image, intensities greater than 2,000 photons/pixel have been set to 2,000 for the upper image and intensities greater than 1,600 photons/pixel have been set to 1,600 for the lower image. To improve contrast of the anisotropy image, anisotropies higher than 0.35 and lower than 0.21 have been set to 0.35 and 0.21, respectively. Regions of high intensity in the emission-intensity image are circled with red dashed lines, and the identical regions in the anisotropy image are circled with black dashed lines. Scale bar represents 10 μm for the upper images, and 2 μm for the lower images.
(B) For the cell in (A), steady-state anisotropy was calculated at regions corresponding to GFP-CBD both at the UCV and in the cytosol, and the mean anisotropy at both localizations is plotted. Mean anisotropy at the UCV and in the cytosol are 0.266 ± 0.003 (n = 58, ± SEM) and 0.286 ± 0.007 (n = 30, ± SEM), respectively.
(C) Steady-state GFP fluorescence emission intensity images (left) and anisotropy images (right) for a cell expressing CBD-GFP. The same presentation is used as in (A). To improve contrast of the fluorescence emission image, intensities greater than 850 photons/pixel have been set to 850 for the upper image and intensities greater than 450 photons/pixel have been set to 450 for the lower image. To improve contrast of the anisotropy image, anisotropies higher than 0.37 and lower than 0.25 have been set to 0.37 and 0.25, respectively. Scale bar represents 10 μm for the upper images, and 2 μm for the lower images.
(D) For the cell in (C), steady-state anisotropy was calculated at regions corresponding to CBD-GFP both at the UCV and in the cytosol, and the mean anisotropy at both localizations is plotted. Mean anisotropy at the UCV and in the cytosol are 0.296 ± 0.019 (n = 15, ± SEM) and 0.269 ± 0.011 (n = 30, ± SEM), respectively.