As a possible alternative to having two button widgets (Overlay1, Overlay2), what about adding the functionality to select multiple overlays in the Open Dialogue listing the files. Something along the lines of either selecting multiple datasets (holding shift or control) or multiple apply and set buttons (one row below) to allow the second overlay to be active. I suppose you would need some indicator to the left of the file names to indicate that they are currently selected. Likely would need a clear button to deselect all overlays.

For Slider bar adjusting (usually) t-value, perhaps a second one immediately next to the first one, allowing specification of independent t-values for each overlay. Each slider would have it's own color spectrum, thus allowing you to set the colors if you dichotomized the color spectrum, you could set positive to one color for overlay1 and another color in overlay2.