Image alignment

See attached PDF for a guide with example images

1. Open images you wish to align and find the equivalent z-plane by looking for the same cells in each image. I look for distinct nuclei in the DAPI channel.
2. Find at least one clear DAPI puncta in all images, mark with the Point Tool in ImageJ and measure the X, Y, and Slice positions.
3. Subtract: X₁ – X_n , Y₁ – Y_n. If your images have a pixels/micron scale, the calculated X and Y values must be multiplied by the scale. For the slice position, take the values from the largest one to find how many slices to add to the “front” of the image. Then subtract the lowest new total from the new total of slices for each image to find the number of slices to remove from the “back”. This leaves images with all the same number of slices allowing you to merge the channels from any cycle. 
4. To shift the images in X and Y go to: Image→Transform→Translate and input the calculated values. Before shifting, I recommend first increasing the canvas size by 2x the largest X and Y values so that none of the image leaves the frame, which causes data to be lost.
5. To shift in Z go to: Image→Stacks→Add Slice or Delete Slice. When adding slices se the Z-position to 1 and select “Prepend” so the new slice will become the first in the stack. When deleting go to the last Z-position.
6. For automated image processing, fill out Alignment_Template.xlsx. Save “CSV” sheet as CSV, and import into ImageJ as “Results”. Open and run 4i_Alignment macro (code in attached txt file).