Orca flash4.0 v2 scmos camera

Microscopy imaging was done using fully automated Olympus IX81 or IX83 inverted microscope systems (Olympus, Japan), equipped with a ×100 NA1.3 oil immersion, phase contrast objective, an ORCA-flash 4.0 v2 sCMOS camera (Hamamatsu, Japan), an automated stage controller (Marzhauser Wetzlar, Germany), shutter, and laser-based autofocus system (Olympus ZDC 1 and 2). Detailed information about the microscopy setup has been described by D’Souza et al. [39 (link)]. Channels on the same PDMS Chip were imaged in parallel, and phase-contrast images of each position were taken every 5 min. The microscopy units and PDMS chip were maintained at room temperature. All experiments were run at a flow rate of 0.1 ml h⁻¹, which ensures nutrients enter the chamber through diffusion. Four biological replicates were performed. These replicates consist of four independent microfluidics channels (two for each of the strains). These channels were connected to one of two independent batch cultures.
The microscopy dataset consists of 200 mother machine channels; 49 channels for the degrader on co-culture, 51 for the degrader on mono-culture, 40 for the cross-feeder on mono-culture and 60 for the cross-feeder on co-culture.

Cells were cultured on glass coverslips, washed with PBS and fixed with 4% PFA. Fixed cells were permeabilized with 0.1% Triton X-100 in TBS for 5′ and moved to 0.2% Dulbecco/BSA. Immunofluorescence stainings were performed as described previously (26 (link)) and were repeated at least three times. The following primary antibodies were used in stainings: anti-E-cadherin (#14472, Cell Signaling Technology, Inc., Danvers, MA, USA), anti-vimentin (#5741, Cell Signaling Technology, Inc., Danvers, MA, USA), and anti-vinculin antibody (1:50) (hVin-1, Sigma, Saint Louis, MO, USA). The following secondary antibodies were used to detect the primary antibodies: Alexa Fluor α-rabbit 488 and α-mouse 568 (Life Technologies™, Carlsbad, CA, USA). Other reagents: Alexa-488- and -647-Phalloidins were used to visualize actin cytoskeleton in 1:200 dilution (Life Technologies™, Carlsbad, CA, USA), DAPI for DNA (Life Technologies™, Carlsbad, CA, USA), and DABCO/Mowiol was used in mounting. The images were acquired with Leica DM6000 upright fluorescence wide field microscope equipped with Hamamatsu Orca-Flash4.0 V2 sCMOS camera.

Confocal microscopy was performed using a Zeiss LSM 710 META confocal microscope equipped with a GaAsP detector with ZEN software (Black Edition), or a spinning-disk system (Marianas; Intelligent Imaging Innovations, Inc.) consisting of an Axio Observer Z1 (Carl Zeiss) equipped with a CSU-WI spinning-disk head (Yokogawa Corporation of America) and an ORCA-Flash4.0 v2 sCMOS camera (Hamamatsu Photonics) with SlideBook 6.0 (Intelligent Imaging Innovations, Inc). Three-dimensional structured illumination microscopy (3D-SIM) was performed using a Zeiss Elyra PS.1 SIM/STORM microscope with a × 63 1.40 numerical aperture (NA) oil objective with Zeiss ZEN software (Black Edition). Adobe Photoshop was used for image analysis, and Z-stack images were generated using ImageJ.