Hey KBaker,
The IMAQ Convert Pixel to Real World VI works well for what you are trying to accomplish. This specific VI transforms pixel coordinates to real-world coordinates, according to the calibration information contained in the image. Calibration information is attached to this image by IMAQ Learn Calibration Template, IMAQ Set Simple Calibration, or IMAQ Set Calibration Info. Once you have set up the calibration for your images, you can simply apply this calibration information to every image from there on out. You can use the two lines you mentioned, or something else you know the real world measurements to, and then set this as your calibration info. Then, in your images you won't have to have that sheet of paper in your image with the lines because you can just apply that information that you took earlier. What happens is that you give a grid descriptor stating the pixel to real world value ratio. This creates a map for the whole image that way you can get the corresponding real world values which allows for the calculation of the real world distance between any two points. A good example program that does this can be found in the NI Example Finder in the following directory: Examples\Vision\2. Functions\Calibration\Perspective Calibration Example.vi.
I hope this helps. Please let me know if you have any further questions regarding this calibration or converting to real world measurements. Thanks and have a great day.
Regards,
DJ
Applications Engineer
National Instruments