Uplfln 100 oil immersion objective

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).

For each sample, 2 μL of cells in suspension were mounted between a glass slide and a 1.5 h glass coverslip, and observed using an inverted IX81 microscope, with the UPLFLN 100× oil immersion objective from Olympus (numerical aperture 1.3), using a fibered Xcite™ Metal-Halide excitation lamp in conjunction with the appropriate excitation filters, dichroic mirrors, and emission filters specific for DAPI/Hoechst, AF488, mCherry or AF647 (4X4MB set, Semrock). Acquisitions were performed with Volocity software (Quorum Technologies) using a sCMOS 2048 × 2048 camera (Hamamatsu ORCA Flash 4, 16 bits/pixel) achieving a final magnification of 64 nm per pixel.