## 2 Materials and Methods  
### Datasets  
Bright-field microscopy images of yeast were produced in house as part of analysis of a novel reporter model. Yeast cells were designed to include synthetic gene networks that allow user-regulated production of fluorescent reporter proteins. Fluorescence time-lapse microscopy was conducted to study the yeast cells under different regulatory regimes using an inverted light microscope with an automated stage and focus control. The images taken at each time point and colony are: 3 bright-field images at different focal planes (for segmentation) and 2 fluorescence images (for expression level quantification). Cells were prepared for microscopy as follows. Single colonies from antibiotic plates were used to inoculate synthetic complete growth medium (with 0.042 g/L adenine hemisulfate and 2% (w/v) glucose added). Resulting overnight cultures (250 RPM shaking, 30 \({}^{\circ}\)C ) were then diluted based on optical density (OD\({}_{600}\) of 0.1, Victor 3V Plate Reader, Perkin Elmer) and returned to the incubator to induce logarithmic phase growth. Following a return to logarithmic phase growth, cultures were further diluted to an OD\({}_{600}\) of 0.07 prior to loading into a microfluidic growth chamber (CellASIC\({}^{\copyright}\) ONIX Y03C microfluidic plate) using a CellASIC\({}^{\copyright}\) ONIX microfluidic control system (EMD Millipore). The standard ONIX software loading protocol (8 psi for 15 s) was used for loading. Upon loading, cells become trapped due to the height of the microfluidic growth chamber designed to allow imaging of cell growth and division in a monolayer. During the course of the time-lapse experiment, chambers were continuously perfused with synthetic complete growth medium (0.042 g/L adenine hemisulfate, 2% (w/v) glucose) at 2 psi, which results in replacement of chamber volume with fresh medium approximately every 13 s.  
Imaging was performed by loading the microfluidics plate onto the stage of an inverted Nikon TiE microscope. Stage movement (X-Y-Z) and imaging was automated using Nikon software (Advanced NIS Elements 3.22.11). To prepare for automation, ten fields of view (FOV) were located manually, each containing one to three cells to limit imaging to one to three colonies per FOV, as were the focal planes for imaging. Thereafter, FOVs were imaged automatically every 10 min. Each FOV was imaged several times per 10 min imaging cycle: a GFP fluorescence image; an mCherry fluorescence image; and bright-field images at 3 focal planes (in focus, 0.6 \(\upmu\)m above focal plane, 1.2 \(\upmu\)m above focal plane). Three bright-field images at and around the center of cells was taken to enable cell segmentation and to counteract focal drift in the Nikon PFS auto-focus system (Perfect Focus 1). Images were captured with a CoolSnap HQ2 CCD camera (Photometrics) and a 60\(\times\) oil immersion objective with a numerical aperture of 1.40 (Nikon Plan Apo VC DIC N2 inf /0.17 WD 0.13). Images were taken at a resolution of 1340 \(\times\) 1092 pixels, bit depth of 214, and were acquired with an exposure time of 200 ms. The complete time-lapse experiment lasted 10 h, or 60 timepoints. The ground-truth dataset resulting from these experiments is as follows. The first 50 time points were used to generate a ground-truth dataset, as within this range colonies did not reach confluence and rogue cells did not pass through the field of view. Each of the 50 time points include 3 bright-field images captured of the same colony. Thus, the 50 ground truths create a dataset of 150 available input images with corresponding labeled true segmentations. To establish ground-truth cell labels, we applied the method described and provided by [PERSON] et al. [18]. Briefly, their method is designed to segment and track yeast cells in a series of images of monolayer colonies acquired by time-lapse microscopy, and is initiated by two GUIs, CTseed and CTtrack, in MATLAB (Mathworks Inc.).