Zdc 2

The detection system was set up on an epi-fluorescence microscope (Olympus IX81). Diode lasers (Toptica Photonics, Munich, Germany) were used for selective fluorescence excitation of GFP, YFP and Cy5/DiD at 488 nm, 514 nm and 640 nm, respectively. A 405 nm diode laser (Toptica Photonics, Munich, Germany) was used for bleaching of GFP/YFP fluorescence. Samples were illuminated in total internal reflection (TIR) configuration (CellTIRF, Olympus) using a 60 x oil immersion objective (NA  =  1.49, APON 60XO TIRF, Olympus, Munich, Germany). After appropriate filtering using standard filter sets, fluorescence was imaged onto a CCD camera (Orca-R2, Hamamatsu, Japan). Samples were mounted on an x-y-stage (CMR-STG-MHIX2-motorized table; Märzhäuser, Germany) and scanning of larger areas was supported by a laser-guided automated focus-hold system (ZDC-2; Olympus). For FRAP experiments single patterns were photobleached with a laser pulse (405 nm) applied for 100 ms. Recovery images were recorded at indicated time intervals. FRAP images were analyzed using the Multimeasure plugin of ImageJ [27] (link). Data were normalized by the pre-bleach image and curve fitting was done using Graphpad Prism. Resulting FRAP curves were plotted based on the standard error of the mean (SEM) and fitted using a bi-exponential equation. Kinetic FRAP parameters were directly obtained from curve fitting.

The setup of microfluidics experiments has been described in detail previously [16 (link), 29 (link)]. Cell growth and behavior was imaged within chambers measuring 60–120 × 60 × 0.56 μm (length × width × height). In these chambers, cells could adhere to the glass surface and experienced TR medium with 0.1% alginate that diffused into the lateral flow channels (0.1 ml h⁻¹). Microscopy imaging was performed using an IX83 inverted microscope system with automated stage controller (Marzhauser Wetzlar), shutter, and laser-based autofocus system (Olympus ZDC 2). Chambers were imaged in parallel on the same PDMS chip, capturing phase-contrast images of each position every 8–10 min. Images were acquired using an UPLFLN 100× oil immersion objective (Olympus) and an ORCA-flash 4.0 v2 or v4 sCMOS cameras (Hamamatsu, Japan). For image acquisition, the CellSens 1.18 and higher software package (Olympus) were used. The microscopy units and PDMS chip were maintained at 25 °C using a cellVivo microscope incubation system (Pecon GmbH).

E. coli BL21(DE3) cells were grown overnight in LB medium and subsequently diluted 1000 times in EZ rich medium (Teknova) with 0.2% glucose. The growth media was supplemented with the appropriate antibiotics. When the cell culture reached an OD600 of 0.3, eGFP expression was induced with IPTG at a final concentration of 200 μM for 30 minutes at 37 °C.
For imaging, 3 μL of the bacterial culture was transferred to a microscope coverslip and covered by an agarose pad [90 (link), 91 (link)].
Imaging was done on an Olympus IX83 inverted microscope with an Olympus UAPON 100x NA 1.49 TIRF oil immersion objective. The excitation light (514 nm, Coherent) was coupled into the objective via the ET—442/514/561 Laser Triple band set (69904, Chroma) and the fluorescence was collected on a electron multiplying charge-coupled CCD camera (512x512 pixel, C9100-13, Hamamatsu). The focal position was held constant by the Olympus ZDC2 and images on multiple positions were collected automatically using Olympus’ CellSens software.