Live-Cell Time-Lapse Imaging of Bdellovibrio bacteriovorus Predation on E. coli

Abstract

Source: Makowski, Ł., et al. Live-Cell Imaging of the Life Cycle of Bacterial Predator Bdellovibrio bacteriovorus using Time-Lapse Fluorescence Microscopy. J. Vis. Exp. (2020)

This video demonstrates time-lapse fluorescence imaging of Bdellovibrio bacteriovorus preying on immobilized E. coli beneath an agarose pad, enabling real-time visualization of intracellular replication and progeny release for studying bacterial predation and host-pathogen interactions.

Protocol

1. Conduction of time-lapse fluorescence microscopy

1. Place the 35 mm dish in the Petri dish holder such that it will not move during the course of the experiment. Place one drop of immersion oil (1.518 refractive index) onto the objective as well as the bottom of the 35 mm dish.
2. Mount the holder onto the stage of the inverted microscope in the microscope chamber.
3. Set up the options in the microscope control software to collect multiple images at multiple stage positions over time.
   1. Set the magnification (100x) and polychromic lens (GFP (green fluorescent protein)/mCherry). Using an eyepiece and brightfield (BF), find the focal plane and open the point list manager.
   2. Select at least 10 positions of interest by moving the stage and storing the coordinates for each position in the microscope software by clicking the Mark point button. Avoid positions located close to each other to prevent photobleaching and phototoxicity. Turn the camera valve from the eyepiece to the monitor and calibrate each point by setting the focal plane and clicking the Replace point button in the point list manager.
   3. Open the experiment designer and set all experimental parameters in each tab as follows:
      1. Unmark the Z-stacking box.
      2. Choose fluorescent channels and set the optimal illumination settings. Here, the following settings were used: for the mCherry channel, mCherry filter sets (EX575/25; EM625/45), 50% intensity, and 200 ms exposure time; for the GFP channel, GFP filter set (EX475/28; EM525/48), 50% intensity, and 80 ms; and for the BF images, POL channel, 5% intensity, and 50 ms.
      3. Select intervals between acquiring images and the total time of the experiment. Here, 5 min intervals were performed over the 10 h period of the experiment.
      4. Enable focus maintenance for the chosen positions (for the system used here, by marking the Maintain focus with UltimateFocus check box).
      5. Select the data folder in which the image files should be automatically saved.
      6. Recheck all settings in the microscope software control, then start the time-lapse experiment.
4. After the first hour of the experiment, check that all stage positions are still in focus. Adjust the focus during time intervals between image acquisition, if needed.
5. When the time-lapse experiment is finished, remove the Petri dish and utilize the 35 mm dish with an agarose pad according to the biosafety protocol.
   NOTE: All substeps after step 1.2 are specific for DeltaVision Elite users. Researchers using other systems need to adjust the settings according to individual systems.

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
CertifiedMolecular Biology Agarose	BIO-RAD	161-3100	Low fluorescence agarose for agarose pad
Fiji	ImageJ	https://imagej.net/Fiji	Open source image processing package
Glass Bottom Dish 35 mm	ibidi	81218-200	Uncoated glass
Microscope	GE	DeltaVision Elite	Microtiter Stage, ultimate focus laser module, DV Elite CoolSnap HQ2 Camera, SSI assembly FP DV, kit obj. Oly 100x oil 1.4 NA, prism Nomarski 100x LWD DIC, ENV ctrl IX71 uTiter opaQ 240 V
Start SoftWoRx	GE		Manufacturer-supplied imaging software