# Temporal reassignment and correspondence evaluation with quality control for time-course imaging of 3D cell culture

## TL;DR

TRACE-QC is a tool that improves the accuracy of time-lapse imaging in 3D cell cultures by correcting mislabeling and displacement issues.

## Contribution

TRACE-QC introduces a Procrustes-based method for correcting mislabeling and displacement in 3D cell culture imaging without experimental changes.

## Key findings

- TRACE-QC accurately reorders and aligns spheroid positions across time points.
- Validation with simulated data confirmed the method's accuracy and robustness.
- The tool revealed and corrected frequent displacement in tumor spheroid imaging.

## Abstract

Longitudinal imaging of 3D cell cultures like tumor organoids and spheroids offers crucial insights into cancer progression and treatment. However, spatial displacement during time-course imaging, caused by matrix detachment or experimental artifacts, can confound analyses. We present TRACE-QC, an application of the Procrustes technique to evaluate data integrity and rectify mislabeling in longitudinal imaging of 3D cell culture. Our algorithm integrates permutation-based optimization with Procrustes analysis. By using X and Y coordinates of images, it accurately reorders, matches, and aligns object positions across time points, correcting for global well rotations and translations, along with local spheroid movements. Validation with simulated data confirmed its accuracy and robustness. Applied to longitudinal imaging of tumor spheroids, our algorithm revealed frequent displacement among the spheroids between time points and corrected many mislabeled images. This computationally efficient and adaptable method needs no experimental adjustments and presents a readily accessible solution for data quality control.

•TRACE-QC is a tool that enhances reliability of spheroid-based assays•Corrects spheroid displacement in 3D cell cultures based on image metadata•Ensures data integrity for longitudinal imaging

TRACE-QC is a tool that enhances reliability of spheroid-based assays

Corrects spheroid displacement in 3D cell cultures based on image metadata

Ensures data integrity for longitudinal imaging

Three-dimensional (3D) in vitro models, such as tumor organoids and spheroids embedded in an extracellular matrix, are increasingly vital for studying normal and disease biology, including drug responses. A key advantage of these models is that imaging platforms can perform continuous longitudinal imaging to follow phenotypic changes. However, common issues in 3D techniques, such as matrix shifts during experimental setup or image capture, can introduce technical artifacts that affect downstream analyses. We sought to develop an automated analytical approach for post-experimental assessment or correction of technical artifacts.

Cramer et al. present TRACE-QC, a method for quality control of time-course imaging in 3D cell cultures. Using iterative Procrustes analysis, it reorders and aligns spheroid positions, corrects mislabeled images, and improves data integrity. TRACE-QC enhances assay reliability and interpretability without experimental modifications or parameter tuning.