This dataset contains the confocal fluorescence microscopy images of nucleus, actin and mitochondria, where each clear image corresponds to 6 out-of-focus images with different degree of blurring. Images acquired below and above the optimal focal plane are blurry and out-of-focus images. In detail, scans were acquired in z-stack of 15 layers spanning the depth (8.4 μm) with 0.6 μm between each slice, z = 7 is the optimal focal plane, z = 1–6 are below the focal plane, z = 8–15 are above the focal plane. We make layers from z = 4 to z = 10 publicly available. Their visual variations in 3-dimensional structure can be negligible. In the end, datasets of actin and nucleus contain 100 in-focus images and 600 out-of-focus images respectively, dataset of mitochondria contain 97 in-focus images and 582 out-of-focus images. Each image is composed of 1024 × 1024 pixels in 8-bit JPG format.