I would suggest two ways of storing overlays in the image file:
1- Make the overlay part of the image content using IMAQ Merge Overlay and then use the regular IMAQ WriteFile to save the flattened image. This will allow you to write BMP, TIFF, JPEG or PNG files.
2- Use IMAQ Write Image and Vision Info to save the image as well as the overlay information to file. When you use this VI, the overlay information is saved in a separate chunk of the PNG file. You need to use IMAQ Read Image and Vision Info to read the image as well as the overlay back from the file.
Hope this will help,
Nicolas