Shifts in image registration can affect colocalization results. (A and B) Images of 100-nm Tetra-Speck beads (Invitrogen; mounted in glycerol) that fluoresce multiple colors including red and green, collected with a microscope (model 80i; Nikon) and camera (ORCA R2; Hamamatsu Photonics) using a Plan-Apochromat 100x NA 1.4 oil objective lens and MetaMorph software. One image of the beads was collected using a filter set for green fluorescence (FITC) and a second image of the beads was collected using a filter set for red fluorescence (TRITC); all other microscope optics were the same between the two images. The two images were pseudo-colored and merged using MetaMorph software. The scatter plots (generated in MetaMorph) display the correlation between the intensity values of the red and green pixels in the images. (A) The merged image, showing a registration shift of several pixels between the red and green images. 10 sets of images were collected to determine that the shift is repeatable, and therefore most likely caused by the filter sets (not depicted). The correlation coefficient for these red and green images is only 0.72, even though the red and green images represent the exact same beads. Bar = 1 µm. (B) The same images as in A, after correction for the shift in registration using MetaMorph image processing software. The correlation coefficient increased to 0.97 after the correction.