CellProfiler 3.0: Next-generation image processing for biology

Abstract

CellProfiler has enabled the scientific research community to create flexible, modular image analysis pipelines since its release in 2005. Here, we describe CellProfiler 3.0, a new version of the software supporting both whole-volume and plane-wise analysis of three-dimensional (3D) image stacks, increasingly common in biomedical research. CellProfiler's infrastructure is greatly improved, and we provide a protocol for cloud-based, large-scale image processing. New plugins enable running pretrained deep learning models on images. Designed by and for biologists, CellProfiler equips researchers with powerful computational tools via a well-documented user interface, empowering biologists in all fields to create quantitative, reproducible image analysis workflows.

Fig 1. Volumetric processing for 3D images of DNA-stained nuclei of hiPSCs using CellProfiler 3.0.

Images are from the Allen Institute for Cell Science, Seattle, and available from the Broad Bioimage Benchmark Collection (https://data.broadinstitute.org/bbbc/BBBC034/). (A) Original 3D image of nuclei monolayer prior to analysis. (B) Evaluation of CellProfiler 3.0 performance in comparison to the MorphoLibJ plugin in Fiji software. Both were compared to manually annotated ground truth using CellProfiler’s MeasureImageOverlap module. (C) Selected CellProfiler 3.0 image processing modules used for hiPSC nucleus segmentation. Figure labels: RH (“RemoveHoles”), EorS Features (“EnhanceOrSuppressFeatures”). (D) Ground truth obtained by manual annotation of each Z-slice using GIMP software. (E) Image processing done using Fiji’s MorphoLibJ plugin (macro code is presented in S1 Table). 3D, three-dimensional; hiPSC, human induced pluripotent stem cell.

Fig 2. Examples of 3D image segmentation produced by CellProfiler 3.0, across two experimental systems and two sets of synthesized images.

Three focal planes shown for each. Raw images (left) and CellProfiler outputs (right) showing nuclei of mouse embryo blastocyst (A), mouse trophoblast stem cells (B), and synthetic images of HL60 cell lines (C) and (D). More information about segmentation steps used for these images can be found in S2–S5 Figs. (E) Comparison of the segmentation accuracy of CellProfiler 3.0 and Fiji’s plugin MorphoLibJ, based on the Rand index of the processed image and its ground truth (out of a total of 1.0). Object accuracy comparisons of these same images may be found in S6 Fig and S3 File. 3D, three-dimensional; hiPSC, human induced pluripotent stem cell.

Fig 3. Segmentation steps for the quantification of transcripts per cell within a 3D blastocyst.

Images were captured of a mouse embryo blastocyst cell membrane stained with WGA and FISH for GAPDH transcripts. (A) Original 3D image of blastocyst cell membrane prior to analysis. (B) CellProfiler 3.0 image processing modules used for membrane image processing. Figure labels: RH (“RemoveHoles”), Close (“Closing”), Erode (“Erosion”), Mask (“MaskImage”), Math (“ImageMath”), EorS Features (“EnhanceOrSuppressFeatures”). (C) Nuclei after segmentation by CellProfiler, as viewed in Fiji. (D) Segmentation of cells after setting nuclei as seeds by CellProfiler, as viewed in Fiji. (E) Segmentation of GAPDH transcript foci using CellProfiler, as viewed in Fiji. (F) Examples of analysis that can be done by CellProfiler: (top) cell volume relative nucleus volume, (middle) GAPDH transcript quantity in each cell using CellProfiler’s “RelateObjects” module, (bottom) number of GAPDH transcripts in Z-plane (bin size = 2.5 μm). The underlying measurements may be downloaded as S1 File. Images were provided by Javier Frias Aldeguer and Nicolas Rivron from Hubrecht Institute, Netherlands, and are available from the Broad Bioimage Benchmark Collection (https://data.broadinstitute.org/bbbc/BBBC032/). 3D, three-dimensional; FISH, fluorescent in situ hybridization; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; WGA, wheat germ agglutinin.

Fig 4. Segmentation and analysis of 3D hiPSC images using CellProfiler 3.0.

DNA channel showing nuclei (A), CellMaskDeepRed channel showing membrane (B), and GFP channel showing beta-actin (C) at the center (left) and edge (right) of the hiPSC colony. (D) Various measurements obtained from the samples are shown; note that cells touching the edge of each image are excluded from this analysis. The underlying measurements may be downloaded as S2 File. Images are from the Allen Institute for Cell Science, Seattle, and are available from the Broad Bioimage Benchmark Collection (https://data.broadinstitute.org/bbbc/BBBC034/). 3D, three-dimensional; GFP, green fluorescent protein; hiPSC, human induced pluripotent stem cell.