A1r ti2 confocal microscope

For transmission electron microscopy analysis, purified carboxysome were first fixed with glutaraldehyde (4% in PBS buffer) for 30 min at room temperature, washed three time with PBS, then adsorbed for 1 min to a carbon-coated grid. The samples were stained with 1.0% uranyl acetate in deionized water. The grids were imaged using 80 kv accelerating voltage using a Hitachi H7500 transmission electron microscope. For imaging using fluorescent microscope, E. coli cells transformed with either pZS-23641-CbbL-mCherry or pZS-23641-CbbL(C)-mCherry were cultured, induced for carboxysome expression, then collected by centrifugation at 5,000 g for 10 min. Cells were washed three times with PBS buffer, then fixed with paraformaldehyde (4% in PBS buffer) for 30 min at room temperature. Cells were then washed, resuspended in PBS, loaded on a glass slide, then imaged using Nikon A1R-Ti-2 confocal microscope. Texas red channel was excited at 595 nm and imaged at 620 nm. The percentage of localization of fluorescence signal is analyzed using ImageJ^{41 (link)}.

RNAscope assay was done using the Hiplex12 fluorescent assay kit for mouse (Catalog # 324443, Advanced Cell Diagnostics, Hayward, CA, USA) per manufacturer’s instructions. Positive and negative control probes were run in parallel to test probes to ensure proper hybridization and imaging conditions were attained in our experiments. Confocal images were captured using a Nikon A1R-Ti2 confocal microscope. Z-series stack with 6 images per stack was captured at a step size of 1 μm. Images were scanned using a 512 × 512 pixel format, scan lines were averaged twice, and frames were scanned 3 times. Acquisition parameters [i.e., gain, offset, photomultiplier tube (PMT) setting] were held constant for experiments. Colocalization counts were made using QuPath software from Advanced Cell Diagnostics. Cell boundaries were detected automatically based on DAPI staining. The fidelity of each cell boundary was confirmed by manual inspection. The number of fluorescent spots in each channel (that corresponds to individual mRNA molecules) per cell were extracted and used for colocalization counting. Data from more than 2 nonconsecutive sections from 2 mice were pooled.