Spectrax led

A plug containing spheroplasts was deposited on a KOH-cleaned coverslip. Spheroplasts were imaged with a Nikon Ti2-E microscope with a 100× CFI Plan Apo Lambda Oil objective with an NA of 1.45 and SpectraX LED (Lumencor) illumination system using the channels phase contrast, cyan (CFP filter cube λex/λbs/λem = 426–446/455/460–500 nm), yellow (triple bandpass filter λem = 465/25–545/30–630/60 nm) and red (the same triple bandpass filter). The imaging protocol was composed of a single time-point, using a 2 μm z stack with 200 nm z-slices.
For imaging chromosomes after lysing the spheroplasts, a nucleoid-containing plug was incubated in 2 mL buffer A (50 mM Tris-HC pH 8, 50 mM NaCl, 1 mM EDTA pH 8.0, 5% glycerol) at 4 ⁰C for 1 h. The plug was transferred to 2 mL imaging buffer (50 mM Tris-HC pH 8, 50 mM NaCl, 1 mM EDTA pH 8.0, 5% glycerol, 3.5 mM MgCl₂, 1 mM DTT, 500 nM Sytox Orange) and incubated for 15 min. Then the plug was deposited on a KOH-cleaned coverslip and 30 μL imaging buffer was added onto the plug to prevent drying. The plug was imaged using an Andor Spinning Disk Confocal microscope with a 100× oil immersion objective, 20% 561 laser, filters, 250x gain, and 10 ms exposure. The imaging protocol resulted in 30 μm z-stacks with 250 nm z-slices and was repeated at 15 distinct XY positions.

The fluorescence measurements were performed on a fully motorized inverted wide field microscope (Nikon Ti-E or Ti-2) equipped with an environmental box which was pre-heated to 30 °C and maintained at this temperature during the whole cultivation time. The microfluidic device was mounted on the microscope using the in-house made holder, allowing an addition of the magnet holder on the top of the microfluidic device.^{31 (link)}The individual compartments were imaged for a 15 h period (10 h every 30 min, 5 h every 1 h) by brightfield and fluorescence microscopy through a 10× objective (Nikon Plan Fluor). Lumencor Spectra X LED was used as a light source for fluorescence excitation (25% of the maximum light intensity) with corresponding optical filters and dichroic mirrors (riboflavin: cyan LED, 475/28 excitation filter, 495 dichroic, 525/50 emission filter; mKate2: green LED, 549/15 excitation filter, 562 dichroic, 593/40 emission filter). The fluorescence and bright field images were recorded by a Hamamatsu Orca Flash 4 camera (exposure times: 20 ms bright field, 100 ms fluorescence). The microscope was controlled with the NIKON NIS-Elements Advanced Research software. The time-lapse images were acquired using the Nikon Perfect Focus System.