This dataset was created for the online microscopy course given by the Live Cell Imaging core facility, Karolinska Institutet, Sweden. Images for this dataset were acquired with the goal of demonstrating the effect of uneven illumination or uneven focus when stitching tiles. The images in all the folders with img2 in their name were acquired with the NIS Elements software to cover 15 consecutive fields of view of the sample. The sample consists of a 16 μm cryostat section of mouse kidney stained with a combination of fluorescent dyes. Alexa Fluor® 488 wheat germ agglutinin, a green-fluorescent lectin, was used to label elements of the glomeruli and convoluted tubules. The filamentous actin prevalent in glomeruli and the brush border were stained with red-fluorescent Alexa Fluor® 568 phalloidin ((Molecular probes, F24630). Finally, the nuclei were counterstained with the blue-fluorescent DNA stain DAPI.The samples were mounted in ProLong® antifade mounting medium. Tiles were acquired with 1%, 5% or 10% overlap (named 1pc, 5pc and 10pc respectively). The images were either kept as separated images (sep) or stitched together into a single image using the NIS Elements software. The images in the uneven_focus folder correspond to the same sample and tiled images as above but additionally, z-stacks of 10, 50 and 100 um were acquired (z-step of 2.5 um). The laser power and exposure time were adjusted to have no saturation in the images. The image was acquired on a Nikon Ti2 inverted microscope using a 10x air objective (NA 0.45), 477 excitation wavelength, 515/30 emission filter and pentaband DM (441/30; 511/26; 593/37; 684/34; 817/66). The camera is a sCMOS Kinetix (Photometrics), pixel size 6.5 um resulting in undersampling. In order to produce separate tiff files and simplify the usage of stitching algorithms, a custom made script found here was developed in FIJI to split the tiled image into its individual tiles (also found zipped in img2_sep_files.zip). Configuration files were also added to the different folders to be used with the BASIC algorithm and the scripts shared in the workshop repository.