Cy5 filter set

Fluorescence measurements were performed on a Nikon TiE inverted microscope driven by the NIS-elements software package (Nikon). The microscope was equipped with an Andor Neo 5.5 sCMOS 12 bit camera (Oxford Instruments), a 40 × 0.75 NA Super Fluor objective, a 60 × 1.4 NA oil-immersion apochromatic objective (Nikon), a perfect-focus mechanism (Nikon), and EGFP and Cy3 TE-series optical filter sets (Chroma), as well Cy5 filter set (Semrock).

Droplets were generated as previously described (Eyer et al., 2017 (link)), and the emulsion was directly injected into the 2D observation chamber. After chamber filling was complete, the chamber was gently closed and mounted onto an inverted fluorescence microscope (Ti Eclipse, Nikon). Two neodymium magnets (BZX082, K and J Magnetics) were placed on each side of the chamber during observation to hold the bead lines in place. Excitation light was provided by a LED source (SOLA light engine, Lumencor Inc). Fluorescence for the specific channels were recorded using appropriate band pass filters (GFP and TRITC filter sets, Nikon, and Cy5 filter set, Semrock) and camera settings (Orca Flash 4, Hamamatsu) at room temperature (25° C) and ambient oxygen concentration. Images were acquired using a 10x objective (NA 0.45). An array of 10 × 10 images were acquired for each experiment, every 7.5 min in all channels over 37.5 min (five measurements total).