How do I take a derivative of data I am receiving from a camera that measures the location of two particles?

[NapDynamite]

If you're goal is to measure the speed at which the 'dot' is moving, why don't you is"IMAQ Optical Flow" functions.
An alternative would be to get x and y coordinates of energy center of the dot, from previous and present iterations (shift register), measure distance and then divide from frame rate.
For example if, for instance, in a 680x480 image size, the dot moved from 150 to 300 pixels with fps of 30. This means the dot moved with 50 pixels per second. Rookie concept but worth looking into. I would still recommend that you use optical flow.

[gnickell555]

I never managed to get the built in derivative function to work but I did get what I wanted by just taking the differences of x values between two different loops and dividing that by the difference in time (dx/dt). Thank you all very much for the help!