DeLTA 2.0: A deep learning pipeline for quantifying single-cell spatial and temporal dynamics

Improvements in microscopy software and hardware have dramatically increased the pace of image acquisition, making analysis a major bottleneck in generating quantitative, single-cell data. Although tools for segmenting and tracking bacteria within time-lapse images exist, most require human input, are specialized to the experimental set up, or lack accuracy. Here, we introduce DeLTA 2.0, a purely Python workflow that can rapidly and accurately analyze single cells on two-dimensional surfaces to quantify gene expression and cell growth. The algorithm uses deep convolutional neural networks to extract single-cell information from time-lapse images, requiring no human input after training. DeLTA 2.0 retains all the functionality of the original version, which was optimized for bacteria growing in the mother machine microfluidic device, but extends results to two-dimensional growth environments. Two-dimensional environments represent an important class of data because they are more straightforward to implement experimentally, they offer the potential for studies using co-cultures of cells, and they can be used to quantify spatial effects and multi-generational phenomena. However, segmentation and tracking are significantly more challenging tasks in two-dimensions due to exponential increases in the number of cells that must be tracked. To showcase this new functionality, we analyze mixed populations of antibiotic resistant and susceptible cells, and also track pole age and growth rate across generations. In addition to the two-dimensional capabilities, we also introduce several major improvements to the code that increase accessibility, including the ability to accept many standard microscopy file formats and arbitrary image sizes as inputs. DeLTA 2.0 is rapid, with run times of less than 10 minutes for complete movies with hundreds of cells, and is highly accurate, with error rates around 1%, making it a powerful tool for analyzing time-lapse microscopy data. Author SummaryTime-lapse microscopy can generate large image datasets which track single-cell properties like gene expression or growth rate over time. Deep learning tools are very useful for analyzing these data and can identify the location of cells and track their position over time. In this work, we introduce a new version of our Deep Learning for Time-lapse Analysis (DeLTA) software, which includes the ability to robustly segment and track bacteria that are growing in two dimensions, such as on agarose pads or within microfluidic environments. This capability is essential for experiments where spatial and positional effects are important, such as conditions with microbial co-cultures, cell-to-cell interactions, or spatial patterning. The software also tracks pole age and can be used to analyze replicative aging. These new features join other improvements, such as the ability to work directly with many common microscope file formats. DeLTA 2.0 can reliably track hundreds of cells with low error rates, making it an ideal tool for high throughput analysis of microscopy data.