Time-Lapse Imaging of Chromosome Segregation in Fluorescently Labeled Bacteria

Begin with a nutrient-rich agarose pad on a metal frame containing bacteria expressing fluorescently tagged chromosome origins and fluorescent microspheres.

The agarose pad supports bacterial attachment and mobility, and fluorescent microspheres serve as a reference to track chromosome segregation.

Add immersion oil to the objective lens in the pre-heated incubation chamber of an inverted widefield microscope.

Place the sample over the lens, then focus on a region containing multiple bacteria and at least one fluorescent microsphere.

Initiate time-lapse imaging and capture both phase-contrast and fluorescence images.

Fluorescently tagged origin of replication appears as a bright cluster near one end of the cell.

During division, the origin duplicates; one cluster remains at the original end while the other cluster migrates to the newly formed cell end.

This setup allows tracking of chromosome segregation over multiple divisions and can be adapted to visualize similar dynamic processes in other bacteria, including pathogenic species during host interactions.

To carry out time-lapse microscopy, switch on the microscope and start the microscope control software. Select the correct objective a