Example dataset that can be used for testing the EASI-FISH (Expansion-Assisted Iterative Fluorescence in situ hybridization) data analysis pipeline (https://github.com/JaneliaSciComp/multifish). Raw image data (.czi) and metadata (.mvl) are provided here. Image data was collected using a Zeiss Lightsheet Z.1 microscope. A 20× water-immersion objective (RI=1.33) was used for imaging with 1× zoom. Single-side illumination was used to reduce light exposure and imaging time. Overlap between tiles was set to 8%. Voxel size (µm): 0.23 x 0.23 x 0.42 (x, y, z). This dataset can be used with EASI-FISH example data (multi-round FISH, round 5) (https://doi.org/10.25378/janelia.13622828) for registration testing. LHA3_R3_medium (32GB): Two image tiles are included in a single CZI file, with 4 channels (3 FISH channels and 1 DAPI channel). Channel1: 488 (Gpr83), channel 2: 669 (Slc17a6), channel 3: 405 (DAPI), channel 4: 546 (Bdnf) and image dimension per tile: 1920 x 1920 x 591 (x, y, z). LHA3_R3_small (12GB): Two image tiles are included in a single CZI file, with 2 channels (1 FISH channel and 1 DAPI channel). Channel1: 488 (Gpr83), channel 2: 405 (DAPI) and image dimension per tile: 1920 x 1920 x 422 (x, y, z). LHA3_R3_tiny (<1GB):Two image tiles are included in two separate CZI files (LHA3_R3_tiny_V01.czi and LHA3_R3_tiny_V02.czi), with 2 channels (1 FISH channel and 1 DAPI channel). Channel1: 488 (Gpr83), channel 2: 405 (DAPI). Please note that this set is for software testing only and the output from this set may not reflect the actual performance of the pipeline, which is optimized for large datasets.