Axio zoom v16 microscope

We measured the fitness difference $s$ between wild type and mutant cells using the colliding colonies assay (Korolev et al., 2012 (link); Gralka et al., 2016b (link)). Briefly, a mutant clone was first isolated and then grown independently of the wild type overnight. In the wild type, the plasmid was maintained by adding 10 μg/ml doxycycline to the overnight culture. After growth overnight, cultures were diluted 1:10, grown for about 1.5 hr, and then washed twice in PBS to remove residual doxycycline. 1 μl droplet of each strain were spotted on agar plates about 2 mm apart. After drying, colonies were grown for 3 days and then imaged under the a Zeiss Axiozoom v16 microscope. The resulting images were used to estimate fitness differences by fitting a circle onto the mutant-wild type interface. The results are shown in Figure 2—figure supplement 1: without doxycyline, mutants have a 20–25% advantage over the wild type. Both strains have equal growth rate around ≈0.35 μg/ml, and the mutants grow more slowly than the wild type at higher concentration of doxycycline.
For the growth rate measurements in Figure 2, colonies were grown from single cells on both rough and smooth plates in a temperature-controlled growth chamber and imaged overnight on a Zeiss Axiozoom v16 microscope. The resulting time lapse movies were binarized and the colony areas extracted.

Brightfield embryonic imaging was performed on a Leica M80 microscope with MC170 HD camera (Leica). Wide-field fluorescence imaging was performed on a Zeiss AXIO Zoom V16 microscope (EMS3/SyCoP3, Zeiss) and ORCA-Flash 4.0 C1140-22C camera (Hamamatsu). Embryonic confocal imaging was performed on a Leica TCS SP8 Lightning confocal with Leica DMI8 microscope, 40x/1.3 water immersion objective and HyD detectors using the 405, 488 and 552 laser lines and Leica LAS X software (Leica). Samples were mounted in 0.8% low-melt agarose on glass-bottom dishes with a coverslip. The images were deconvolved using the Lightning module (Leica LAS X Lightning, Leica). All time-lapse imaging was performed using a resonant scanner with 16 times averaging. Maximum intensity projections and time lapse images were recorded using Imaris release 9.9.1 (Bitplane). Brain confocal imaging was performed on a Leica TCS SP8 Lightning confocal with Leica DMI8 microscope, 10x/0.4 air immersion objective and HyD detectors using the 552 and 638 laser lines and Leica LAS X software (Leica). Samples were cleared in glycerol and mounted on glass slides with coverslips. Images were prepared using Imaris Viewer version 9.9.1 (Bitplane), Photoshop release 23.4.2 (Adobe) and Illustrator release 26.4.1 (Adobe).