For image substraction you first need to store the image in a image buffer and then use a operation to subtract them. see example script.
For the Auto trashold, the standard trashold of the Image assistand has some predified auto trashold options, if you wish to modify this, you will need to generate for example LabVIEW code and program it your self.
The same is true for overlaying images, this is not a feature in Image Assistand and needs to be programmed in code.
Met vriendelijke groet / Kind regards,
Karsten
Applications Engineer
National Instruments