Correlation procedure based on fiducials. (A) Merge of GFP and RFP channels of Rvs167-EGFP/Abp1-mCherry expressed in S. cerevisiae. The patch of interest is highlighted by the circle. Inset shows the Rvs167-EGFP signal of that patch, boxed as for centroid fitting. A cross marks the resulting centroid position. (B) Blue FluoSpheres (365 nm/415 nm) channel. Fiducials selected for correlation are marked with yellow circles and numbers. (C) Same fiducials as in B present on merged slices of the section surface from a tomogram of the cell of interest are assigned to the corresponding fluorescent spots in the FM image (marked by yellow circles and numbers). White circle and guidelines correspond to A and B after applying the optimal transform, calculated using the coordinates of the selected fiducials. (D) Slice of high magnification tomogram showing the endocytic invagination. Cross marks the centroid coordinates of the fluorescent patch transformed into tomogram coordinates. The inner circle corresponds to a probability radius of 50% (33 nm), outer circle to 80% (89 nm). Bars: (A–C) 1 µm; (D) 50 nm.