New answers tagged sprite
I've recently encountered this problem and I found a tool called Stitches. It allows you to drag in images and organize them in a few different way. This app will generate a sprite-sheet as well as a CSS document and supports LESS as well.
Yes, it could be done with a script, surely. But it might take longer to write and test the script than to do it manually. Since it looks like you're doing a transform, then blending between them. Would the blend tool work for partial automation?
Photoshops your best bet, although it doesn't like it if you use Save for Web on humongous images, you can just do a straight save if you want to keep your alpha channel. You don't say what the sprites are being used for but most game development API's support targeting one colour (say 255,255,0) as an alpha channel.
Adobe's Photoshop supports something like 300,000 pixel canvas's. It has a "Save for Web" feature that allows you to save a PNG24 file (which supports your alpha transparency). It too has all the functions you are looking for.
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