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 and the correct mirrors and filter to acquire phase contrast images as well as images of green-fluorescent, red-fluorescent, or yellow-fluorescent proteins. Add a drop of high-quality immersion oil onto the lens of the objective and to the bottom of the sample pre-incubated at 32 degrees Celsius.

With the hole side towards the objective, place the metal frame with the sample onto the microscope stage, then fasten the sample securely in the stage holder. Focus on the cells by moving the stage in the Z-direction closer to the objective. When the oil on the objective and sample make contact, move the stage in the X/Y-direction.

Switch to the Metamorph software and open the Acquire tool. Select Phase Contrast in the Setting dropdown list and set the Exposure Time to 100 milliseconds. Click Show Live, Bring Cell into Focal Plane, and move stage in X/Y-direction until multiple single cells are visible in the field of view.

Ensure that at least one fluorescent microsphere is in the region of view to later align the acquired images. Next, open the Multi-Dimensional Acquisition wizard of the microscope control software to set up a time-lapse experiment that allows the microscope to acquire images at multiple wavelengths and stage positions if required.

In the Main tab, activate time-lapse and Multiple Wavelengths. Additional tabs will appear on the left side of the window. Click on the Saving tab and Select Directory to select an empty folder on the computer hard drive to save the acquired images, then activate Increment Base Name If File Exists to make sure the consecutive datasets do not overwrite earlier ones. Give the experiment a name with date and the strain name or title of the experiment.

Click on the time-lapse tab to adjust the time-lapse parameters, then set the Time Interval to 20 minutes and set Duration to 24 hours. The number of time points will change automatically. Now, click on the Wavelengths tab.

Select the number of wavelengths to acquire for each image at each time point by changing the number. Select Allow Separate Hardware Memorized AF Position for Each Wavelength. Click the First Wavelength tab from the top.

In the Illumination dropdown list, select Phase Contrast. Select 100 milliseconds for exposure, and in the Acquire dropdown list, select Every Time Point. Deactivate Auto Expose in the dropdown list by selecting Never, and select Every Acquisition in the dropdown list for Auto Focus.

Set the exposure for every wavelength as just demonstrated using the following parameters. Then, to acquire images from multiple stage positions, in the Main tab, activate Multiple Stage Positions. Click on the Stage tab and click the Live button to look at the field of view.

Move the stage in the X/Y-direction until an ROI is in the field of view. Save the X and Y-coordinates in the Stage tab by clicking the plus sign. Move the stage again in the X/Y-direction until a new ROI is found and save the coordinates again by clicking the plus sign.

Continue until the desired number of regions are saved. Check once more that the cells are in focus by clicking on the different saved X and Y-positions, and start the hardware autofocus by clicking AFC hold to keep the saved Z-position constant over the course of the experiment. Start the time-lapse recording in the microscope control software's Multi-Dimensional Acquisition wizard by clicking Acquire.

Check that the cells are still in focus after the first few time points in the time-lapse recordings to maximize the quality of the images and refocus if required.