NIH Image Tutorial: Measuring the Eye Open NIH Image. Use File>Open to open the stack file you created in the previous section. In Analyze>Set Scale enter 4.200 pixels per mm. Set your parameters to measure in Analyze>Options. Click on the Area and Perimeter/Length boxes to leave them checked. Open Analyze>Show Results. A box will appear with the measurement options that you have chosen in step 3, but no measurements. As you measure the area of the eye for each image in your stack, that measurement will appear in the Results box. This way you will know if you have accidently measured an area twice. If you have measured the image twice, use Analyze>Delete Measuremet. Use Analyze>Redo Measurement if you are not satified with the measurement you took. Use the zoom tool to zoom into the eye on the first image in the stack. Use the trace tool to select the eye by dragging the cursor around the perimeter of the eye. Analyze>Measure the area you have chosen in slice 1. Do this only once; if you do it more than once you will get more than one entry in the resulting data table. If you want to do it over, use Analyze>Redo Measurement. Use Analyze>Delete Measurement to remove the extra measurement before preceding. Edit>Deselect before going on to the next slice. Go to the next slice using Stacks>Next Slice. Repeat steps 7-10 on each of the slices in the stack. Click on the Results Box to make it active then use File>Save As to save your data. Click on the Measurement button in the Save As pop-up box. - Page 3 of 4 -