The cells were allowed to adapt to growth in the device for at least two additional hours before imaging. Time-lapse images in experiments performed at 34°C were acquired on a Nikon Eclipse Ti-inverted microscope equipped with a 60× Plan Apo oil objective (numerical aperture, 1.4; Nikon), an Orca R2 charge-coupled device (CCD) camera (Hamamatsu), an automated xy stage (Ludl), and a SOLA light engine light-emitting diode (LED) excitation source (Lumencor). The microscope was surrounded by a temperature-controlled enclosure. The following filter sets were used for acquisition: YFP (Semrock YFP-2427A), CFP (Semrock CFP-2432A), and red fluorescent protein (RFP; Semrock mCherry-A). Automated time-lapse acquisition was controlled using custom MATLAB 2011a (MathWorks) scripts interfacing with μManager 1.4. Images were taken every 10 min, and focal drift was corrected via the Nikon PerfectFocus system and periodically recalibrated using z-stacks on a sacrificial position. Images in the RFP (the cell segmentation marker) were acquired at full camera resolution (1344 × 1024 pixels) to improve segmentation, while CFP and YFP images were acquired using 2 × 2 binning to reduce measurement noise. Short exposure times (typically 200 to 300 ms) and low illumination intensities (<30% of maximum illumination power) were used to minimize the effects of photobleaching.

Time-lapse images in subsequent experiments at 30°C were acquired on a Zeiss Axiovert 200M microscope equipped with a Plan-Apochromat 40×/1.3 Oil Ph3 Objective, a CCD camera (Hamamatsu C4742-98-24ERG), and a fluorescence excitation LED illumination source (SOLA SE II, Lumencor). Filters with the following specifications were used: for YFP [excitation (Ex), 500/24; dichroic (Di), 520; emission (Em), 542/27], CFP (Ex, 438/24; Di, 458; Em, 483/32), and RFP (Ex, 586/20; Di, 605; Em, 647/57). All filters were from Semrock. The variation in fluorescence intensity illumination across the field of view was less than 10% in all channels. The microscope setup was controlled using custom software on MATLAB 2013a (MathWorks) interfacing with μManager 1.4. Images were acquired every 5 min. Focal drift was corrected at each acquisition step via a custom autofocus routine, which acquires phase-contrast images at planes above and below the previously determined optimal autofocus plane and estimates the image plane with maximal contrast. Once the new focal plane was determined, images in the YFP, CFP, and RFP channels were acquired at full camera resolution (1344 × 1024 pixels). Again, short exposure times (typically 200 to 300 ms) and low illumination intensities (<15% of maximum illumination power) were used to minimize the effects of photobleaching.

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