Piskel
Check a preview of your animation in real time as you draw. Adjust the frame delay on the fly. Several export modes supported. Animated GIFs for sharing, spritesheet PNG/ZIP for bigger projects etc. All the code is open-source and available on GitHub.
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Pixen
Pixen is a professional pixel art editor designed for working with low-resolution raster art, such as those 8-bit sprites found in old-school video games. Pixen packs all the tools pixel artists need in an intuitive, native interface including support for high zoom levels, animation editing, color palettes, and a lot more.
Leverage Pixen's wide array of tools when composing pixel art: Pencil, Eraser, Brightness, Text, Eyedropper, Zoom, Rectangular Selection, Magic Wand, Lasso, Move, Fill, Line, Rectangle, and Ellipse.
Take advantage of the flexibility layers provide when working with complex pixel art. Change opacity, blending modes, and more to compose creative effects.
Use filters to adjust colors, create effects, and post-process your artwork.
With Pixen, you can add and organize commonly-used colors into palettes. Pixen can even import and export color palettes in the Adobe Color Table, Microsoft Palette, JASC Palette, and GIMP Palette formats.
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Pyxel Edit
Pyxel Edit is a pixel art drawing application especially designed for working with tiles. Place tiles to form a level, edit them directly to see how they all work together, then export your tileset and the level data, and load it into your game. Tiles can even be flipped and rotated, still being editable and synced. This feature is inspired by the awesome Pixothello and Cosmigo Pro Motion, but taken one step further. It also supports making animations, and exporting them as sprite sheets or animated GIFs. Pyxel Edit was originally a free program, and the free version is still available. However continued development to make it a more solid and full featured application has taken a lot of the developers time so the next big update will be paid. Initially there will be a discounted beta version available to support continued development. Buyers of the beta will get the normal priced later versions without further cost.
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Aseprite
Aseprite lets you create 2D animations for video games. From sprites, to pixel-art, retro style graphics, and whatever you like about the 8-bit and 16-bit era. It has a color mode, which tell you how many colors the image can handle. All images in the sprite are in one specific mode, you cannot mix RGB images with Indexed images in the same sprite. It contains a set of layers. You can see them in the Timeline. The most important concept here is that there are two kind of layers: the background layer for opaque sprites, and transparent layers. A sprite can contain only one background layer, but several transparent layers. It contains animation frames. Each frame has a duration, i.e. how many milliseconds the frame must be on screen when the animation is being played. Integrate Aseprite in your assets pipeline with the command-line interface (CLI). Store several animations in one texture atlas.
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