Human U2OS cells (out of focus)

Accession number BBBC006 · Version 1

Example images

  • Ground truth: in focus
    Ground truth: in focus
  • Out of focus
    Out of focus
  • Ground truth: foreground
    Ground truth: foreground

Biological application

Since robust foreground/background separation and segmentation of cellular objects (i.e., identification of which pixels below to which objects) strongly depends on image quality, focus artifacts are detrimental to data quality. This image set provides examples of in- and out-of-focus HCS images which can be used for validation of focus metrics.


Images were acquired from one 384-well microplate containing U2OS cells stained with Hoechst 33342 markers (to label nuclei) were imaged with an exposure of 15 and 1000 ms for Hoechst and phalloidin respectively, at 20x magnification, 2x binning, and 2 sites per well. For each site, the optimal focus was found using laser auto-focusing to find the well bottom. The automated microscope was then programmed to collect a z-stack of 32 image sets (z = 16 at the optimal focal plane, 15 images above the focal plane, 16 below) with 2 μm between slices. Each image is 696 x 520 pixels in 16-bit TIF format, LZW compression. Each image filename includes either 'w1' to denote Hoechst images or 'w2' to denote phalloidin images. (798 MB) (801 MB) (803 MB) (804 MB) (806 MB) (807 MB) (809 MB) (809 MB) (810 MB) (810 MB) (809 MB) (809 MB) (808 MB) (806 MB) (805 MB) (803 MB) (800 MB) (798 MB) (795 MB) (793 MB) (791 MB) (791 MB) (791 MB) (793 MB) (795 MB) (797 MB) (799 MB) (801 MB) (803 MB) (804 MB) (805 MB) (805 MB) (806 MB) (806 MB)

Ground truth C

For each of the 768 fields of view (384 wells, 2 fields of view per well), an automated algorithm used the image at optimal focus (z = 16) to identify and count the nuclei. The counts are provided as a CSV file: (66 kB)

Example rows from the file:

Image_Count_Nuclei Image_FileName_OrigDAPI Image_Metadata_Site Image_Metadata_Well

Ground truth F O

The images were manually examined by an expert to classify which focal planes corresponded to in- and out-fous images. Planes between z = 11 - 23 are considered ground truth as in-focus images.

For in-focus images, the pixels belonging to the nuclei were automatically identified as foreground and segmented. These ground truth images are 8-bit grayscale PNG files, with each segmented nuclei receiving a unique integer label. If the segmentation is not needed, the foreground can be thresholded with all pixel values greater than 0. (9.8 MB)


The cells were cultured by Sigrun Gustafsdottir and the images were acquired by Tom Hasaka. Mark Bray identified the foreground.

Published results using this image set

Nucleus counts (32 z-step increments, with 2304 fields of view each) Citation
BBBC006_results_bray.csv (2.4 MB) Bray et al., J. Biomol Screen, 2011

Recommended citation

"We used the image set BBBC006v1 from the Broad Bioimage Benchmark Collection [Ljosa et al., Nature Methods, 2012]."


CC0 To the extent possible under law, Anne Carpenter has waived all copyright and related or neighboring rights to out-of-focus U2OS images and ground truth. This work is published from: United States.

This page last updated 2018-01-31