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1. Quantification of Polar Localization and Dynamics of Dot/Icm Components
NOTE: The following steps are designed for images with 0.129 µm per pixel that were acquired with 2 x 2 binning.
- Quantification of polarity for sfGFP (superfolder green fluorescent protein) fusion proteins (Figure 1)
- Adjust the image contrast so the bacteria are clearly visible. Use the region tool to place a 0.25 x 1.3 µm2 rectangle starting at the pole and extending into the cytoplasm. The rectangle must remain precisely within the bacterial borders.
- Mark at least 200 bacteria and use the Region to Mask button to create masks for the regions of interest. Under Mask Statistics and Mask Scope, choose Object. Then, under Features and Intensity, mark Mean Intensity and Variance.
- Export the data and calculate the polarity scores of each bacterium as the ratio between the variance to the mean intensity.
- For high-throughput applications, use a phase objective and an appropriate condenser setup to acquire images with the phase and 488 nm channels. Make sure to choose fields of view where the bacteria are fully separated.
- Adjust the phase channel contrast of the image to a level where the bacteria are clearly visible. Open the dual channel image, launch the Create Segment Mask window and change the channel to phase.
- Adjust an appropriate threshold and remove small objects with the Define Objects button. Under Refine Mask, choose Remove Edges Objects, and later separate masks of bacteria that are adjacent to each other.
- Calculate the polarity scores of the signal in the 488 channel for each cell as was previously described in steps 1.1.2–1.1.3.
- Quantification of dynamics for sfGFP fusion proteins (Figure 2)
NOTE: Dynamics are defined as changes in intensity over time, and the following steps are designed for short imaging periods (i.e., several minutes). Add to the pad the appropriate supplements if longer imaging periods are desired.- In the Image Capture window, mark Timelapse, enter the time of intervals in the interval box, and enter 2 in the # of Time Points box. Acquire two successive images of Legionella pneumophila expressing the fluorescent protein of interest.
- Adjust the image contrast until the cells are clearly visible. Follow Figure 2A and the descriptions below to place three different masks. Use the region tool to place a 0.25 x 0.25 µm square in the middle of at least 400 cells.
- Use the Region to Mask button to create a mask (mask 1) of the squares of interest. Create a new empty mask (mask 2) and use the pixel tool or the polygon tool to mark the entire cell area of at least 25 random cells, which will be used to calculate fluorescence bleaching. Create a new empty mask (mask 3) and use the large brush tool to mark areas between the cells, which will be used for background subtraction.
- Under Mask Statistics and Mask Scope, choose Object for mask 1 and mask 2. Then, under Features and Intensity, choose Mean Intensity and export the data of the two masks. For mask 3, export the mean intensity of the entire mask.
- Calculate the change in fluorescence intensity for each object in mask 1 using the following formulas:
where mask 1 is a 0.25 µm × 0.25 µm square placed at the cell center, mask 2 covers the whole cell, mask 3 is the background between cells, t1 is the mean intensity in the first time point, and t2 is the mean intensity in the second time point.
