Three-dimensional alignment and merging of confocal microscopy stacks

We describe an efficient, robust, automated method for image alignment and merging of translated, rotated and flipped confocal microscopy stacks. The samples are captured in both directions (top and bottom) to increase the SNR of the individual slices. We identify the overlapping region of the two stacks by using a variable depth Maximum Intensity Projection (MIP) in the z dimension. For each depth tested, the MIP images gives an estimate of the angle of rotation between the stacks and the shifts in the x and y directions using the Fourier Shift property in 2D. We use the estimated rotation angle, shifts in the x and y direction and align the images in the z direction. A linear blending technique based on a sigmoidal function is used to maximize the information from the stacks and combine them. We get maximum information gain as we combine stacks obtained from both directions.