4.4 Confocal microscopy
All images shown in this paper were captured by Nikon A1 LFOVT. To assay
compartment formation (Figures 2C and 2D) and light responses (Figures
3B, 3C and 3E) for a long period of time, we used an LB agar pad to fix
1-5 μL overnight E. coli under a microscope. The solid pad layer
was made from 400 μL 1% LB agarose, on a glass coverslip-bottomed (20
mm in diameter) 35 mm Petri dish (D35-20-1-N). To observe bacterial
morphology and compartment localization pattern (Supplementary Figure
2-4), recruitment of enzymes into compartments (not shown), and other
short period observation, we used glass slides to fix them. All images
captured were under a 100X oil immersed lens. 488 nm laser was used both
for the observation of phase module distribution and the inducing light
signal to trigger POI recruitment. 561 nm laser was used for the
observation of POI recruitment. ProLong Live Antifade Reagent (P36975,
Invitrogen) was used to avoid photo-bleaching of mCherry (1:50
dilution). Intense light induction was performed using region of
interest (ROI) to guide 488 nm lasers, 3% lasted for 8 seconds (Figures
3B and 3E), while weak light induction was conducted under 2% laser
intensity for 6 seconds. FRAP assays were carried out using slightly
higher laser power with ROI as a single pixel.