Dietrich Kohlheyer

Karina Ruzaeva, Jan-Christopher Cohrs, Keitaro Kasahara et al.

Cell tracking is an essential tool in live-cell imaging to determine single-cell features, such as division patterns or elongation rates. Unlike in common multiple object tracking, in microbial live-cell experiments cells are growing, moving, and dividing over time, to form cell colonies that are densely packed in mono-layer structures. With increasing cell numbers, following the precise cell-cell associations correctly over many generations becomes more and more challenging, due to the massively increasing number of possible associations. To tackle this challenge, we propose a fast parameter-free cell tracking approach, which consists of activity-prioritized nearest neighbor assignment of growing cells and a combinatorial solver that assigns splitting mother cells to their daughters. As input for the tracking, Omnipose is utilized for instance segmentation. Unlike conventional nearest-neighbor-based tracking approaches, the assignment steps of our proposed method are based on a Gaussian activity-based metric, predicting the cell-specific migration probability, thereby limiting the number of erroneous assignments. In addition to being a building block for cell tracking, the proposed activity map is a standalone tracking-free metric for indicating cell activity. Finally, we perform a quantitative analysis of the tracking accuracy for different frame rates, to inform life scientists about a suitable (in terms of tracking performance) choice of the frame rate for their cultivation experiments, when cell tracks are the desired key outcome.

Johannes Seiffarth, Keitaro Kasahara, Michelle Bund et al.

Live-cell imaging (LCI) technology enables the detailed spatio-temporal characterization of living cells at the single-cell level, which is critical for advancing research in the life sciences, from biomedical applications to bioprocessing. High-throughput setups with tens to hundreds of parallel cell cultivations offer the potential for robust and reproducible insights. However, these insights are obscured by the large amount of LCI data recorded per experiment. Recent advances in state-of-the-art deep learning methods for cell segmentation and tracking now enable the automated analysis of such large data volumes, offering unprecedented opportunities to systematically study single-cell dynamics. The next key challenge lies in integrating these powerful tools into accessible, flexible, and user-friendly workflows that support routine application in biological research. In this work, we present acia-workflows, a platform that combines three key components: (1) the Automated live-Cell Imaging Analysis (acia) Python library, which supports the modular design of image analysis pipelines offering eight deep learning segmentation and tracking approaches; (2) workflows that assemble the image analysis pipeline, its software dependencies, documentation, and visualizations into a single Jupyter Notebook, leading to accessible, reproducible and scalable analysis workflows; and (3) a collection of application workflows showcasing the analysis and customization capabilities in real-world applications. Specifically, we present three workflows to investigate various types of microfluidic LCI experiments ranging from growth rate comparisons to precise, minute-resolution quantitative analyses of individual dynamic cells responses to changing oxygen conditions. Our collection of more than ten application workflows is open source and publicly available at https://github.com/JuBiotech/acia-workflows.

Johannes Seiffarth, Matthias Pesch, Lukas Scholtes et al.

High-throughput microfluidic live-cell imaging generates rich single-cell data. Yet semi-automated procedures for locating regions of interest (RoIs), each containing one cell population, and removing surrounding microfluidic structures from recorded images, scale with the number of RoIs. This prevents real-time image analysis and delays time-to-insight by hours to days. We introduce the Design-Aware and Real-Time capable (DART) paradigm for microfluidic cultivation chips, which aligns the CAD blueprint with the physical chip and thereby enables throughput-independent localization of all RoIs and fully automated image processing across diverse RoI geometries and chip layouts. DART establishes this alignment through embedded fiducial markers and deep-learning-based marker detection. We validate DART using the Swiss Army Knife chip, which combines eight structurally distinct RoI designs across 1164 RoI locations. DART localizes all RoIs in five minutes, removes microfluidic structures from raw microscopy images in 40 ms, and performs fully automated image analysis, including cell segmentation, in under 1.1 s per image. Together, these capabilities establish DART as an end-to-end hardware-software paradigm with real-time-capable analysis that paves the way toward closed-loop and outcome-driven smart microscopy.

Nils Friederich, Angelo Jovin Yamachui Sitcheu, Annika Nassal et al.

Microfluidic Live-Cell Imaging (MLCI) generates high-quality data that allows biotechnologists to study cellular growth dynamics in detail. However, obtaining these continuous data over extended periods is challenging, particularly in achieving accurate and consistent real-time event classification at the intersection of imaging and stochastic biology. To address this issue, we introduce the Experiment Automation Pipeline for Event-Driven Microscopy to Smart Microfluidic Single-Cells Analysis (EAP4EMSIG). In particular, we present initial zero-shot results from the real-time segmentation module of our approach. Our findings indicate that among four State-Of-The- Art (SOTA) segmentation methods evaluated, Omnipose delivers the highest Panoptic Quality (PQ) score of 0.9336, while Contour Proposal Network (CPN) achieves the fastest inference time of 185 ms with the second-highest PQ score of 0.8575. Furthermore, we observed that the vision foundation model Segment Anything is unsuitable for this particular use case.

Nils Friederich, Angelo Jovin Yamachui Sitcheu, Annika Nassal et al.

Microfluidic Live-Cell Imaging (MLCI) yields data on microbial cell factories. However, continuous acquisition is challenging as high-throughput experiments often lack real-time insights, delaying responses to stochastic events. We introduce three components in the Experiment Automation Pipeline for Event-Driven Microscopy to Smart Microfluidic Single-Cell Analysis (EAP4EMSIG): a fast, accurate Multi-Layer Perceptron (MLP)-based autofocusing method predicting the focus offset, an evaluation of real-time segmentation methods and a real-time data analysis dashboard. Our MLP-based autofocusing achieves a Mean Absolute Error (MAE) of 0.105 $μ$m with inference times from 87 ms. Among eleven evaluated Deep Learning (DL) segmentation methods, Cellpose reached a Panoptic Quality (PQ) of 93.36 %, while a distance-based method was fastest (121 ms, Panoptic Quality 93.02 %).