Imaging Morphological Changes in Bacteria Using Live-Cell Fluorescence Microscopy

Begin with a bacterial culture immobilized under an agarose slab in a glass-bottom dish.

The bacterial cell membranes are labeled with a fluorescent dye.

The bacteria are also engineered to express an antisense RNA that targets a specific mRNA, inhibiting production of an essential cell division protein.

Add a drop of immersion oil to the objective of the inverted fluorescence microscope.

Place the dish into a metal housing and secure it on the microscope stage.

Raise the objective until the oil contacts the dish, then focus on the cells.

Use imaging software to set Z-stacks for capturing images at multiple depths.

Begin acquiring fluorescence time-lapse images.

As the cells respond to the antisense RNA expression, they fail to divide and develop swollen shapes.

The membrane organization becomes disrupted, resulting in uneven and focal fluorescence that indicates defective cell division.

To image the sample, use the coarse adjustment focus knob to make sure that the objective is fully lowered before initializing the microscope within the microscope software. Place a drop of 1.517 refractive index oil into the 100X oil immersion objective, and transfer the glass-bottom dish containing the sam