Fluorescence Live-cell Imaging of the Complete Vegetative Cell Cycle of the Slow-growing Social Bacterium Myxococcus xanthus

This method can help answer key questions in the field of bacterial cell biology, such as how do cells replicate their DNA, how do they grow, and how do they divide. The main advantage of this technique is that living bacterial cells can be monitored for at least 24 hours under the microscope and the technique does not require special equipment. Though this method can provide insight into Myxococcus xanthus's growth and division, it can also easily be applied to other slow-growing bacterium.

To begin, re-suspend a single M.xanthus colony in 500 microliters of 1%CTT, supplemented with antibiotics, in a sterile tube and transfer the entire suspension to a 50-milliliter Erlenmeyer flask containing five milliliters of 1%CTT. Prepare 1%agarose microscopy solution containing 0.2%CTT by mixing one gram of agarose with 80 milliliters of TPM buffer and 20 milliliters of 1%CTT medium. Microwave the solution until the agarose is molten.

Fill a Petri dish with approximately 60 milliliters of molten agarose and let it cool down to room temperature. Pre-warm the agarose pad at 32 degrees Celsius for at least 15 minutes prior to use. Next, place a sterile glass coverslip on a plastic or metal frame that has a hole in the middle, then use tape to fix the coverslip to the frame.

Add 10 to 20 microliters of exponentially grown M.xanthus cells onto the coverslip. To add fluorescent microspheres as fiducial markers of the cells, use TPM buffer to dilute the microspheres to 1:100. Shake the bead suspension thoroughly and add five to 10 microliters to the cells.

From the large, pre-warmed 1%agarose pad, cut out a small pad approximately the size of the cover slip and place it on top of the cells. Then place a cover slip on top of the pad to prevent evaporation and to maintain cells in a humid environment. Incubate the microscopy sample at 32 degrees Celsius for 15 to 20 minutes to let the cells attach to the bottom of the agarose pad before time-lapse microscopy recordings.

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. To generate time-lapse movies and perform image alignment, start the image analysis and processing software. Open the images as a stack by clicking Review Multi-Dimensional Data, Select Base File, Select Directory, then open the folder with the multi-dimensional data.

Check the dataset and click View. The dataset will be shown as single images from Time Point One until the end. Activate the wavelength for creating a stack, then select all images that should be in the stack and click Load Images.

Repeat this step for all wavelengths and save completed stacks. Activate the stack of images that needs to be corrected for drift. Open the alignment tool using Apps, Auto Align.

Check Stack as the source for the images and First Plane/Time Point as the reference plane, then select the stack with the Source Stack button and click Apply. When the automatic alignment is complete, save the aligned stack. To generate a movie in MOV or AVI formats, open the Make Movie function via Stack, Make Movie.

Select the time-lapse recordings with the Source Stack button, then select the output format, the frame rate, the number of frames, and click Save. In this time-lapse experiment with motile DK1622 wild type cells, phase contrast images were acquired every five minutes for 24 hours. As expected, cells were motile and predominantly moved in groups.

In phase contrast live cell imaging with non-motile Delta-mgIA cells, the growth and division of individual cells was followed during microcolony formation. When images were acquired every five minutes for 24 hours, it was possible to quantify the interdivision time of 235 plus or minus 50 minutes at single-cell resolution. To investigate whether cells grow normally while tracking yfp-labeled proteins over long periods, M.xanthus cells expressing ParB-YFP were tracked.

ParB-YFP initially formed a single cluster in the subpolar cell region. Shortly before or after cell division, the cluster duplicated, with one cluster remaining at the old cell pole and the second translocating to the new cell pole. In this experiment, non-motile cells expressing FtsZ-gfp showed strong accumulation of FtsZ-gfp at the midcell, which dictates the position of cell division.

FtsZ-gfp formed a cluster at midcell, predominantly in longer cells. Two hours after cell division, FtsZ-gfp accumulated at midcell in the daughter cells. Once mastered this technique can be done routinely on Myxococcus xanthus as well as on any other slow-growing bacterium.

When setting up this procedure for a bacterium, it is important to adjust the composition of the growth medium in the agar plate to meet the specific growth requirements of that bacterium. For any bacterium, it is important to determine the optimal imaging conditions such as exposure time, light intensity, and imaging frequency to avoid phototoxicity. Also, for any fluorescently-labeled protein, the imaging conditions need to be adjusted to avoid phototoxicity and also photobleaching.