Fluorescence Time-Lapse Imaging of the Streptomyces venezuelae Life Cycle

Take a microfluidic plate with a well containing spores of Streptomyces venezuelae, a hyphae-forming bacterium.

Other designated wells contain either nutrient-rich or spent medium.

The spores are derived from a strain expressing fluorescent DivIVA and FtsZ proteins, which localize to hyphal tips and cross-walls, respectively.

Apply immersion oil to the objective lens of an inverted microscope and to the plate’s imaging window.

Mount the plate on the microscope stage and begin time-lapse imaging.

Load the spores into the plate’s culture chamber.

Then, introduce the nutrient-rich medium to initiate spore germination.

As the hyphae grow, DivIVA accumulates at the hyphal tips, while FtsZ forms scattered rings at the cross-walls.

Next, switch to the spent medium. The lack of nutrients in this medium supports sporulation.

In sprouting hyphae, DivIVA foci disappear, and FtsZ reorganizes into a regular ladder of division rings, forming sporulation septa.

Over time, the disappearance of FtsZ fluorescence in the same hyphae confirms completion of sporulation.

Pre-warm the environmental chamber to 30 degrees Celsius in advance.

Then turn on the microscope and the microscope control software. Put a high numerical aperture oil immersion objective into place and confirm that the appropriate filters and diagrammatic mirrors set to acquire differential interference contrast images, along with the images of the yellow florescent and the red fluorescent protein fusions are in place. Place a drop of immersion oil into the objective and to the bottom of the imaging window on the microfluidic plate as well.

Carefully mount the sealed microfluidic device onto the stage of the inverted microscope and secure it into place. Use the embedded position markers to bring the imaging window of the microfluidic culture chamber into focus. Focus on the leftmost part of the first flow chamber labeled A, and then move the stage to trap size five, corresponding to the trap height of 0.7 micrometers.

In the microfluidic software, set the system to load cells from inlet well eight at four psi for 15 seconds. After the process is run, check the cell density in the culture chamber by moving the stage across the imaging window. If no spores were trapped, repeat the cell loading step or alternatively, increase the loading pressure and/or time until the desired cell density of one to 10 spores per imaging window is achieved.

Take care to avoid overloading the culture chamber. Next, start the previously prepared flow program in the control software and allow the microfluidic plate to heat equilibrate for one hour in the microscope stage before starting image acquisition. In the microscope control software, set up a multidimensional acquisition to take multiple images at multiple stage positions over time by first specifying a directory for the automatic saving of the image files.

Next, go to the illumination settings and enter predetermined optimal illumination settings for each specific construct. Then set up a time series to acquire images every 40 minutes for 24 hours. In order to determine stage positions and set the autofocus, scan the culture chamber and store stage positions for each imaging position of interest.

Ensure that the single-stage positions are located far enough apart to minimize photo bleaching and photo toxicity. Once the Z coordinates of the selected stage positions are verified, activate the hardware autofocus. Then start the time-lapse experiment in the microscope control software.

Stop the image acquisition after 24 to 30 hours, or when the hyphae in the region of interest have differentiated into spores. Then stop the flow program in the software and disassemble the microfluidic device. Prepare the used microfluidic plate for short-term storage by removing any remaining medium from the inlet wells, the waste well, and the cell loading well.

Then fill up the used wells of lane A and wells of the unused lanes with sterile PBS. Finally, seal the plate with pear film to prevent it from drying out. And store the plate at four degrees Celsius.