A total of 1 ml culture of the E. coli strain to be imaged was grown from an overnight culture till OD600 = 0.1–0.2. It was then chilled on ice followed by centrifugation at 6000 × g, 4 °C for 1 min to form a cell pellet. Then they were washed with ice-cold 1× PBS twice and resuspended in 100 μl 1× PBS.
In all, 1.5% (w/v) agarose gel was prepared by dissolving agarose in 1× PBS. A few μl cell suspension was sandwiched between a No. 1.5 glass coverslip (VWR) and a thin slab of the agarose gel. The sample was then imaged.
The epifluorescence images were acquired by a Nikon Ti Eclipse microscope (Nikon Instruments, Inc.) using an oil immersion objective (1.46 NA, 100×), which spans an area of ~133 × 133 μm2 for DIC (no filter, autofluorescence) and fluorescence imaging (Ex 480–500 nm, Em 509–547 nm, exposure time 200 ms). The images were acquired using an EMCCD camera (Andor). They were processed using the NIS-Element AR software (Nikon Instruments, Inc.).
In all, 1.5% (w/v) agarose gel was prepared by dissolving agarose in 1× PBS. A few μl cell suspension was sandwiched between a No. 1.5 glass coverslip (VWR) and a thin slab of the agarose gel. The sample was then imaged.
The epifluorescence images were acquired by a Nikon Ti Eclipse microscope (Nikon Instruments, Inc.) using an oil immersion objective (1.46 NA, 100×), which spans an area of ~133 × 133 μm2 for DIC (no filter, autofluorescence) and fluorescence imaging (Ex 480–500 nm, Em 509–547 nm, exposure time 200 ms). The images were acquired using an EMCCD camera (Andor). They were processed using the NIS-Element AR software (Nikon Instruments, Inc.).