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 sample into the metal housing coffin.

Gently slide the dish into the stage clamp, and use the coarse adjustment knob to raise the objective until the oil makes contact with the glass bottom of the dish. Use the eyepiece and the fine adjustment knob to bring the sample into focus, and switch to Camera Mode. In the imaging software on the Resolve 3D window, select the Design/Run Experiment icon.

A new dialog box will appear entitled Design/Run Experiment. Open the Design and Sectioning tabs in the dialog box to set the number of Z stacks and the sample thickness. To measure the thickness of the cells in the sample, incrementally adjust the Z-plane using the up and down arrows in the Resolve 3D dialog box, making where the cells go out-of-focus as the upper and lower limits for the Image Acquisition.

After adjusting the Percent Transmission of the Light Intensity and the duration of the Exposure for the individual selected channels in the Resolve 3D dialog box, click Point List to open the Points List. In the Design and Time Lapse tabs, select the Time Lapse checkbox and enter the time lapse parameters. Select the Visit Point list option, and enter the points to be imaged in the text box, separating the points by commas or hyphens for complete sequences.

Then, edit file names and file locations in the Run tab and select Play in the Begin Experiment dialog box to start the experiment.