An open-source pipeline for longitudinal single-cell tracking and cell-cycle/migration coupling analysis for neurotherapeutic screening.

Overview

1. Department of Diagnostic Radiology and Nuclear Medicine University of Maryland School of Medicine Baltimore Maryland USA
2. Janelia Research Campus Howard Hughes Medical Institute Ashburn Virginia USA

Abstract

Background: Imaging‐based phenotypic assays are widely used in neuroprotection, drug discovery, and neurorepair studies, but long‐term, high‐throughput time‐lapse datasets remain difficult to analyze because of segmentation noise, photobleaching, crowded fields, cell division, and tracking errors. Fluorescent ubiquitination‐based cell cycle indicator (FUCCI) reporters enable live visualization of cell‐cycle phases, but their integration with migration behaviors remains limited. This study aimed to developed an open workflow for tracking cycle and motility.

Methods: We combined Fiji/ImageJ preprocessing, Cellpose‐based deep learning segmentation, and TrackMate‐based cell tracking. FUCCI‐expressing HEK293 cells were imaged using a Tecan Spark Cyto (Männedorf, Switzerland) every 15 min for 24 h. Cellpose models were retrained using manually corrected masks from brightfield and fluorescence images, and TrackMate parameters were optimized using manually curated tracks. Statistical analyses were conducted using Paleontological Statistics (PAST) software (version 4.0, Natural History Museum, University of Oslo, Oslo, Norway).

Results: Iterative cellpose retraining improved segmentation accuracy in both brightfield and fluorescent datasets by reducing false‐positive and false‐negative errors (Brightfield Type I error improvement: t(10) = 33.323, p = 1.400 × 10−11, Brightfield Type II error improvement: t(10) = 16.066, p = 1.805 × 10−8, Fluorescent Type II errors: U = 0, p = 0.00077). TrackMate optimization improved trajectory continuity, especially through adjustment of frame‐to‐frame linking distances (Brightfield: χ 2 (2) = 10, p = 0.00077, Fluorescent: χ 2 (2) = 10, p = 0.00077). The workflow enabled single‐cell and population‐level analysis of migration distance, turning behavior, morphology, division events, and FUCCI‐defined cell‐cycle progression. Cells showed heterogeneous motility, with lower displacement trends during green‐dominant S/G2/M phases than red‐dominant G1 phases.

Conclusion: This workflow links cell‐cycle state with migration dynamics and can be adapted for neuroprotective screening, neural cultures, organoids, and neuron–glia co‐culture studies.

Reproduced under the paper's license (CC BY-NC), from the paper cited above.

Code

The paper links to its data, not to its authors' code: see the Data section.

The paper's code and data availability statement is in the Data section.

Tracing map

A tracing map links a paper to the code its authors published: this paper has none, so it has no map.

Data

Datasets cited

• figshare:31634557, at figshare; found in “DATA AVAILABILITY STATEMENT”

Data availability statement

Codes used in this study can be found in the Supporting Information. Raw image data and tracking file can be found here: https://doi.org/10.6084/m9.figshare.31634557. Other data are available on request from the authors.

Reproduced under the paper's license (CC BY-NC), from the paper cited above.

Versions

The history of this record: each version stored by the harvester or made by a correction of its authors or of the maintainers of its code, and what changed in its facts. The texts of the paper (its abstract, its availability statements) are not part of it; versions that changed only those are not listed.

Version 1, 27 September 2026: the first record

Recorded: type, language, journal, volume, issue, pages, dates, 7 authors, 6 keywords, 2 funders, 27 references.

Cite

This paper

Chandra, N., Yang, M., Wang, H., Janowski, M., Walczak, P., Allier, C., & Liang, Y. (2026). An open-source pipeline for longitudinal single-cell tracking and cell-cycle/migration coupling analysis for neurotherapeutic screening. Neuroprotection (Chichester, England), 4(3), 295-305. https://doi.org/10.1002/nep3.70047

Similar papers

The papers with a page that share the most with this one: the tools found in their code, their categories, datasets, cited references and authors, the rarest counting most.

[1] doi:10.1002/advs.202515913 [code]

Intravital Multimodal Imaging of Human Cortical Organoid Transplantation in a Mouse Model of Chronic Stroke.

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

In common: author Yajie Liang

[2] doi:10.1002/advs.202522762 [code]

Enhancing Maturation of Human Neuromuscular Organoids via Electrical Stimulation.

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

In common: 2 references

[3] doi:10.1038/s41586-026-10391-0 [code]

Cell-type-targeted mitochondrial transplantation rescues cell degeneration.

Journal: Nature

In common: 2 references

[4] doi:10.1186/s40478-026-02415-7 [code]

A standardized framework resolves ambiguity in motor neuron loss across neurodegenerative diseases.

Journal: Acta neuropathologica communications

In common: 2 references

[5] doi:10.1371/journal.pcbi.1014571 [code]

SynAPSeg: A novel dataset and image analysis framework for deep learning-based synapse detection and quantification.

Journal: PLoS computational biology

In common: 2 references

[6] doi:10.1016/j.cell.2026.05.047 [code]

An emergent disease-associated motor neuron state precedes cell death in ALS.

Journal: Cell

In common: 2 references

[7] doi:10.1364/boe.600665 [code]

NeuroSeg-MF: robust neuron segmentation in two-photon Ca<sup>2+</sup> imaging using multi-feature fusion and detection-guided SAM.

Journal: Biomedical optics express

In common: 2 references

[8] doi:10.1038/s41467-026-74320-5 [code]

Spatial architecture of autism pathogenesis reveals mosaic structural disarray during early development.

Journal: Nature communications

In common: 2 references

[9] doi:10.1038/s41467-026-72130-3 [code]

Retinoic acid drives cell fate specification, maturation and retinal regionality in human retinal organoids.

Journal: Nature communications

In common: 2 references

[10] doi:10.1002/advs.202519893 [code]

NeuroSuite for Long-Term Functional and Structural Studies of Air-Liquid Interface Cerebral Organoids.

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

In common: 1 reference

Contribute

The authors of this paper can claim it, correct its record and validate its tracing map, and the maintainers of its code (its owner, or a public member of its organization) correct what it says of their repository; anyone signed in can ask for its removal. Every request goes to OSCR's own machine, which answers it; your account page follows them.

Request its removal

To ask OSCR to remove this record, the copies of its authors' scripts or its tracing map, use the removal request page: signed in, you say who you are, what to remove and why, then review and confirm the request. Published rules decide every request (how).

Discussion: questions and error reports about this paper and its code, from signed-in readers and its authors. It opens with sign-in.

Reproductions: reports from readers who ran the authors' code: what they reproduced, with which environment, commit and data. It opens with sign-in.

Activity: what happens around this paper: new versions of its record, its map's validation, discussions and reproductions. It opens with sign-in.