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). It can be used with EASI-FISH example data (multi-round FISH, round 3 https://doi.org/10.25378/janelia.13622819) for registration testing. LHA3_R5_medium (32GB):Two image tiles are included in a single CZI file, with 4 channels (3 FISH channels and 1 DAPI channel). Channel1: 488 (Gad1), channel 2: 669 (Col25a1), channel 3: 405 (DAPI), channel 4: 546 (Synpr) and image dimension per tile: 1920 x 1920 x 575 (x, y, z).LHA3_R5_small (11GB):Two image tiles are included in a single CZI file, with 2 channels (1 FISH channel and 1 DAPI channel). Channel1: 488 (Gad1), channel 2: 405 (DAPI) and image dimension per tile: 1920 x 1920 x 414 (x, y, z).