Extended Data Fig. 1: Creation of the WTC-11 hiPSC Single-Cell Image Dataset v1 that contains over 200,000 live, high-resolution, 3D cells spanning 25 cellular structures.

The dataset was generated by a microscopy pipeline composed of three main parts; Data Collection, Image Processing and Single-Cell Feature Extraction. a. Data Collection: the sample preparation starts with a vial of frozen gene-edited hiPS cells from a line from the Allen Cell Collection, expressing an endogenous, fluorescently tagged protein representing a particular cellular structure. The cells are cultured in 6-well plates on an automated cell-culture platform. At each passage cells are seeded into optical grade, glass-bottom 96-well plates to create imaging samples. Bright-field overview images of each well are inspected and only wells meeting pre-determined quality controls are passaged from the 6-well plates and imaged from the 96-well plates. The image acquisition of live cells starts with a 12X overview image of each well on a spinning-disk confocal microscope to keep track of the position of each image within each colony. Imaging sessions are conducted using three modes to capture variations in colony area, locations within the colony, and enrich for images with mitotic cells as needed. In mode A, the 12X overview images of colonies are segmented by an automated script to generate sets of coordinates for positions within imageable colonies, located approximately halfway between the colony edge and colony centre. Imageable colonies are those that meet size, morphology, and position-within-a-well criteria. In mode B, the microscope operator adjusts the location of the field of view (FOV) to enrich for mitotic cells via appropriate cell and DNA morphology visible with live bright-field viewing and confirmed by DNA staining (yellow arrows). In mode C, three regions of colonies are imaged, the edge, ridge (just inward from the edge), and centre. The combination of these three imaging modes permitted sampling across all regions of the hiPS cell colonies (Extended Data Fig. 12). Cells were labelled with fluorescent DNA and membrane dyes and then imaged at each pre-selected colony position. Z-stacks were acquired at 120X in four channels, representing the bright-field, cell-membrane dye (magenta), DNA dye (cyan) and the fluorescently tagged cellular structure (grayscale), also shown in (b). Mode A and C panels show Golgi (via sialyltransferase) and microtubules (via alpha-tubulin), respectively. b. Image Processing: The WTC-11 hiPSC Single-Cell Image Dataset v1 consists of a total of 18,100 FOVs curated specifically for successful cell and cellular structure segmentations, which are available for download. An example z-stack is shown. On the left is the maximum intensity projection of all 65 slices with all fluorescent channels combined, in the colours indicated in the panels on the right. “Cutting” the z-stack in half exposes the view of a single slice (slice 32) in the middle of the stack, shown for each individual channel, including the bright-field channel. We applied 3D segmentation algorithms to each of the fluorescent channels to identify boundaries in 3D of the cells via the membrane dye (magenta), the nuclei and mitotic DNA via the DNA dye (cyan), and each of the 25 cellular structures via their fluorescent protein tag (grayscale; Golgi shown here). Resulting 3D segmentations for cell membrane, DNA, and structure channels are also shown as a side view, the xz-cross-section along the yellow dotted line. All segmentation algorithms were developed and performed using the Allen Cell & Structure Segmenter. c. Single Cell Feature Extraction: A total of 215,081 single cells were segmented from the FOVs. Every individual cell was labelled with a unique ID and metadata related to the sample, experiment, and microscopy was collected and associated with each individual cell for future data provenance. Appropriate features were extracted for each cell from the cell, the nucleus or mitotic DNA, and the cellular structure segmentations, including measurements such as the height and volume. These cells, including the images and the segmentations as well as the metadata and features are all available for download. Scale bars are 10 µm unless otherwise noted. d. Number of cells for each cellular structure in the WTC-11 hiPSC Single-Cell Image Dataset v1, sorted by their acquisition order. This table includes all of the various different subsets of the data used throughout the study, including the baseline interphase dataset (excluding outliers, see Methods), mitotic cells, cells within the 8-dimensional sphere (Fig. 3), cells at the edges of colonies (Fig. 4) and cells in early stages of mitosis (m1 and m2, Fig. 5).